Science, Irving K. Barber Faculty of (Okanagan)
Chemistry, Department of (Okanagan)
DSpace
UBCO
Darbyshire, Amanda
Doctor of Philosophy - PhD
University of British Columbia
Fusobacterium nucleatum is a gram-negative bacterium that is ubiquitous to the human oral cavity and has been associated to different pathologies. This thesis focuses on three distinct enzymatic pathways that are involved in F. nucleatum amino acid metabolism. Chapter 2 and 3 focus on serine synthase, a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the reversible reaction of ʟ-cysteine and H₂O to form ʟ-serine and H₂S. The mechanism for this reaction requires an active site aspartic acid residue (D232) to act as a general acid/base catalyst. Hydrophobic residues surrounding D232 increase the basicity of the carboxylate side chain and restrict the range of nucleophilic substrates that can dock into the second binding site. Mutation of these aromatic residues allows for addition of thiol-based nucleophiles to the α-aminoacrylate intermediate of the PLP cofactor, thus allowing serine synthase to act as a potential biocatalytic tool for noncanonical amino acid synthesis. Chapter 4 focuses on lanthionine synthase, another PLP-dependent enzyme, which catalyzes the condensation reaction of ʟ-cysteine with a second equivalent of ʟ-cysteine to produce ʟ,ʟ-lanthionine and H₂S. In this chapter, we utilized the relaxed substrate scope of lanthionine synthase to produce different lanthionine analogs and test potential inhibitors. We also determined that the physiological production of meso-lanthionine in F. nucleatum is likely through the reaction of lanthionine synthase with ʟ-cysteine and D-cysteine and not through an epimerase. Fn1732 in the F. nucleatum genome may act as a racemase on an amino acid other than ʟ,ʟ- lanthionine. Chapter 5 investigates a putative metallochaperone system for lysine 5,6- aminomutase (5,6-LAM). 5,6-LAM is a PLP- and adenosylcobalamin (AdoCbl)-dependent enzyme involved in the second step of the lysine fermentation pathway, catalyzing the conversion of β-ʟ-lysine to erythro-3,5-diaminohexanoate. Like other AdoCbl-dependent enzymes, 5,6-LAM undergoes suicide inactivation during catalysis as a result of the loss of the 5′-deoxyadenosyl moiety and the irreversible oxidation of cob(II)alamin to hydroxocobalamin. We have identified kamB and kamC, two genes responsible for the ATP-dependent reactivation of 5,6-LAM. KamBC is structurally distinct from reactivating factors of other AdoCbl-dependent enzymes, and therefore adds to the list of proteins that have evolved to maintain cellular activity of AdoCbl-dependent enzymes.
Uncovering Distinct Aspects of Amino Acid Metabolism in Fusobacterium nucleatum by Amanda Darbyshire B.Sc., The University of British Columbia (Okanagan), 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) May 2024 ã Amanda Darbyshire ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Uncovering Distinct Aspects of Amino Acid Metabolism in Fusobacterium nucleatum submitted by Amanda Darbyshire in partial fulfillment of the requirements of the degree of Doctor of Philosophy in Biochemistry and Molecular Biology Dr. Kirsten Wolthers (Irving K. Barber Faculty of Science) Supervisor Dr. Frederic Menard (Irving K. Barber Faculty of Science) Supervisory Committee Member Dr. Thu-Thuy Dang (Irving K. Barber Faculty of Science) Supervisory Committee Member Dr. Natalie Strynadka (Faculty of Medicine) University Examiner Dr. Stephen Bearne (Dalhousie University) External Examiner iii Abstract Fusobacterium nucleatum is a gram-negative bacterium that is ubiquitous to the human oral cavity and has been associated to different pathologies. This thesis focuses on three distinct enzymatic pathways that are involved in F. nucleatum amino acid metabolism. Chapter 2 and 3 focus on serine synthase, a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the reversible reaction of L-cysteine and H2O to form L-serine and H2S. The mechanism for this reaction requires an active site aspartic acid residue (D232) to act as a general acid/base catalyst. Hydrophobic residues surrounding D232 increase the basicity of the carboxylate side chain and restrict the range of nucleophilic substrates that can dock into the second binding site. Mutation of these aromatic residues allows for addition of thiol-based nucleophiles to the a-aminoacrylate intermediate of the PLP cofactor, thus allowing serine synthase to act as a potential biocatalytic tool for noncanonical amino acid synthesis. Chapter 4 focuses on lanthionine synthase, another PLP-dependent enzyme, which catalyzes the condensation reaction of L-cysteine with a second equivalent of L-cysteine to produce L,L-lanthionine and H2S. In this chapter, we utilized the relaxed substrate scope of lanthionine synthase to produce different lanthionine analogs and test potential inhibitors. We also determined that the physiological production of meso-lanthionine in F. nucleatum is likely through the reaction of lanthionine synthase with L-cysteine and D-cysteine and not through an epimerase. Fn1732 in the F. nucleatum genome may act as a racemase on an amino acid other than L,L-lanthionine. Chapter 5 investigates a putative metallochaperone system for lysine 5,6-aminomutase (5,6-LAM). 5,6-LAM is a PLP- and adenosylcobalamin (AdoCbl)-dependent enzyme involved in the second step of the lysine fermentation pathway, catalyzing the conversion of b-L-lysine to erythro-3,5-diaminohexanoate. Like other AdoCbl-dependent enzymes, 5,6-LAM undergoes suicide inactivation during catalysis as a result of the loss of the 5′-deoxyadenosyl moiety and the irreversible oxidation of cob(II)alamin to hydroxocobalamin. We have identified kamB and kamC, two genes responsible for the ATP-dependent reactivation of 5,6-LAM. KamBC is structurally distinct from reactivating factors of other AdoCbl-dependent enzymes, and therefore adds to the list of proteins that have evolved to maintain cellular activity of AdoCbl-dependent enzymes. iv Lay Summary Fusobacterium nucleatum is a bacterium that lives in the oral cavity of humans and contributes to the development of periodontal disease. Recent studies have also shown a link between F. nucleatum and various extra-oral diseases such as colorectal cancer. Targeting enzymatic pathways that are vital to the bacterium’s survival will stop or slow its growth, thus improving the health outcomes of people with F. nucleatum-associated infections. This thesis focuses on three key pathways involved in F. nucleatum amino acid metabolism. Given that this bacterium requires the metabolism of amino acids for energy and survival, an understanding of the structure and function of these enzymes involved in these pathways could lead to the development of drug targets for controlling the growth of F. nucleatum. v Preface The results in Chapter 2 have been published and some figures have been reproduced with permission: Darbyshire, A. L., Mothersole, R. G., and Wolthers, K. R. A fold type II PLP-dependent enzyme from Fusobacterium nucleatum functions as a serine synthase and cysteine synthase. Biochemistry 60, 524-536 (2021). The work presented in Chapter 3 has been published and some of the figures have been reproduced with permission: Darbyshire, A. L., and Wolthers, K. R. Expanding the b-substitution reactions of serine synthase through mutagenesis of aromatic active site residues. Archives of Biochemistry and Biophysics 746, 1-10 (2023). Some of the results in Chapter 4 have been published and some figures have been reproduced with permission: Darbyshire, A. L., Mothersole, R. G., and Wolthers, K. R. Biosynthesis of meso-lanthionine in Fusobacterium nucleatum. Archives of Biochemistry and Biophysics 743, 1-12 (2023). Cloning, expression, and purification of lanthionine synthase was performed by Dr. Robert Mothersole, a previous PhD student in the Wolthers group, who also conducted some of the fluorescence experiments described in Section 4.2.10. The results in Chapter 5 have been published and some figures have been reproduced with permission: Darbyshire, A. L., and Wolthers, K. R. Characterization of a structurally distinct ATP-dependent reactivating factor of adenosylcobalamin-dependent lysine 5,6-aminomutase. Biochemistry (2024). Purified ornithine 4,5-aminomutase was prepared by Dr. Caitlyn Makins, a previous PhD student in the Wolthers group. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface ............................................................................................................................................ v Table of Contents ......................................................................................................................... vi List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiv List of Abbreviations ................................................................................................................. xix Acknowledgements ................................................................................................................... xxii Chapter 1: Introduction ............................................................................................................... 1 1.1 Fusobacterium nucleatum .................................................................................................. 1 1.1.1 Fusobacterium nucleatum as an opportunistic pathogen ............................................ 1 1.1.2 F. nucleatum in the oral cavity ................................................................................... 1 1.1.3 Associations to extra-oral diseases ............................................................................. 3 1.1.4 H2S production by F. nucleatum ................................................................................. 5 1.1.5 Pyridoxal 5′-phosphate-dependent enzymes ............................................................... 6 1.1.6 Fold types of PLP-dependent enzymes ....................................................................... 8 1.2 Serine synthase ................................................................................................................... 9 1.2.1 Serine biosynthesis ..................................................................................................... 9 1.2.2 Serine synthase reaction mechanism .......................................................................... 9 1.2.3 The hydrophobic active site of serine synthase ........................................................ 10 1.2.4 Noncanonical amino acid synthesis .......................................................................... 12 1.3 Lanthionine synthase ....................................................................................................... 14 1.3.1 The peptidoglycan layer in F. nucleatum incorporates meso-lanthionine ................ 14 1.3.2 Lanthionine synthase reaction mechanism ............................................................... 15 1.3.3 Lanthionine synthase structure ................................................................................. 16 1.3.4 Fold type II PLP-dependent enzymes exhibit substrate promiscuity ....................... 17 1.3.5 Production of lanthionine analogs ............................................................................ 18 vii 1.3.6 Fn1732 as a potential lanthionine epimerase ............................................................ 19 1.4 Lysine 5,6-aminomutase and KamBC ............................................................................. 20 1.4.1 Lysine fermentation pathway .................................................................................... 20 1.4.2 Adenosylcobalamin-dependent isomerases .............................................................. 22 1.4.3 5,6-LAM reaction mechanism .................................................................................. 24 1.4.4 The “edge-on” conformation in the structure of 5,6-LAM ....................................... 25 1.4.5 Suicide inactivation in AdoCbl-dependent enzymes ................................................ 27 1.4.6 KamBC as a putative metallochaperone system for 5,6-LAM ................................. 27 1.5 Research Objectives ......................................................................................................... 28 Chapter 2: Serine Synthase ........................................................................................................ 29 2.1 Chapter Summary ............................................................................................................ 29 2.2 Experimental Procedures ................................................................................................. 29 2.2.1 Materials ................................................................................................................... 29 2.2.2 Cloning and expression of serine synthase ............................................................... 30 2.2.3 Enzyme purification of serine synthase .................................................................... 31 2.2.4 Construction of serine synthase variant D232N ....................................................... 32 2.2.5 UV-visible spectroscopy ........................................................................................... 32 2.2.6 Steady-state kinetic assays measuring H2S production ............................................ 33 2.2.7 Steady-state kinetic assays measuring L-cysteine production ................................... 33 2.2.8 Steady-state kinetic assays measuring pyruvate production ..................................... 34 2.2.9 Stopped-flow spectroscopy ....................................................................................... 34 2.2.10 Fluorescence spectroscopy ....................................................................................... 35 2.3 Results .............................................................................................................................. 36 2.3.1 Properties of purified serine synthase ....................................................................... 36 2.3.2 Steady-state kinetic analysis of H2S production ....................................................... 40 2.3.3 Steady-state kinetic analysis of L-cysteine production ............................................. 41 2.3.4 Steady-state kinetic analysis of pyruvate production ............................................... 42 2.3.5 Pre-steady-state kinetic analysis with L-cysteine ...................................................... 44 2.3.6 Pre-steady-state kinetic analysis with L-serine .......................................................... 50 viii 2.3.7 Fluorescence analysis with L-cysteine and L-serine .................................................. 53 2.4 Discussion ........................................................................................................................ 56 2.4.1 Serine synthase also functions as a cysteine synthase .............................................. 56 2.4.2 Active site comparison to CBS and OASS ............................................................... 58 2.4.3 D232 acts as an acid/base catalyst in the forward and reverse reactions of SS ........ 59 2.4.4 D232 stabilizes the more chemically reactive ketoenamine PLP tautomer .............. 60 2.4.5 The hydrophobic active site of SS may result in the stabilization of the quinonoid species ....................................................................................................................... 61 2.4.6 Detection of the quinonoid species in other fold type II PLP-dependent enzymes .. 62 2.5 Concluding Remarks ........................................................................................................ 63 Chapter 3: Serine Synthase Variants ........................................................................................ 65 3.1 Chapter Summary ............................................................................................................ 65 3.2 Experimental Procedures ................................................................................................. 66 3.2.1 Materials ................................................................................................................... 66 3.2.2 Construction of SS variants ...................................................................................... 66 3.2.3 UV-visible spectroscopy ........................................................................................... 67 3.2.4 Steady-state kinetic assays measuring H2S production ............................................ 68 3.2.5 Steady-state kinetic assays measuring L-cysteine production ................................... 68 3.2.6 Stopped-flow spectroscopy ....................................................................................... 69 3.2.7 Fluorescence spectroscopy ....................................................................................... 69 3.2.8 Absorbance assays with other nucleophiles .............................................................. 69 3.3 Results .............................................................................................................................. 70 3.3.1 Properties of SS variants ........................................................................................... 70 3.3.2 Steady-state kinetic analysis of H2S production ....................................................... 73 3.3.3 Steady-state kinetic analysis of L-cysteine production ............................................. 76 3.3.4 Pre-steady-state kinetic analysis with L-cysteine ...................................................... 77 3.3.5 Pre-steady-state kinetic analysis with L-serine .......................................................... 83 3.3.6 Fluorescence analysis with L-cysteine and L-serine .................................................. 89 3.3.7 Reaction of SS variants with other nucleophiles ...................................................... 92 ix 3.4 Discussion ........................................................................................................................ 96 3.4.1 Mutation of hydrophobic residues in SS affects the acid/base activity of D232 ...... 96 3.4.2 The positioning of active site residues in SS may affect catalytic performance ...... 97 3.4.3 The hydrophobic active site cluster of SS favours the forward reaction with L-cysteine .................................................................................................................................. 99 3.4.4 SS variants have biocatalytic potential in the synthesis of noncanonical amino acids .................................................................................................................................. 99 3.5 Concluding Remarks ...................................................................................................... 100 Chapter 4: Lanthionine Synthase ............................................................................................ 102 4.1 Chapter Summary .......................................................................................................... 102 4.2 Experimental Procedures ............................................................................................... 102 4.2.1 Materials ................................................................................................................. 102 4.2.2 Cloning and expression of lanthionine synthase and Fn1732 ................................. 103 4.2.3 Enzyme purification of lanthionine synthase and Fn1732 ...................................... 104 4.2.4 Construction of lanthionine synthase variant S224A ............................................. 105 4.2.5 UV-visible spectroscopy ......................................................................................... 105 4.2.6 Steady-state kinetic assays measuring lanthionine production ............................... 105 4.2.7 High-performance liquid chromatography (HPLC) ............................................... 106 4.2.8 Stopped-flow spectroscopy ..................................................................................... 106 4.2.9 Absorbance assays with potential inhibitors of lanthionine synthase ..................... 107 4.2.10 Fluorescence spectroscopy ..................................................................................... 108 4.2.11 Steady-state kinetic inhibition assays ..................................................................... 108 4.2.12 Substrate docking simulations ................................................................................ 109 4.3 Results ............................................................................................................................ 109 4.3.1 Properties of purified Fn1732 ................................................................................. 109 4.3.2 UV-visible spectroscopy ......................................................................................... 110 4.3.3 Steady-state kinetic analysis of lanthionine production ......................................... 111 4.3.4 HPLC analysis of lanthionine products .................................................................. 113 4.3.5 Pre-steady-state kinetic analysis with OAS and L-cysteine or D-cysteine .............. 115 4.3.6 Spectral analysis of potential inhibitors .................................................................. 120 x 4.3.7 Fluorescence inhibition analysis ............................................................................. 122 4.3.8 Steady-state kinetic inhibition analysis ................................................................... 124 4.3.9 Substrate docking simulations ................................................................................ 125 4.4 Discussion ...................................................................................................................... 126 4.4.1 Cysteine derivatives can react with LS to form different lanthionine products ..... 126 4.4.2 Lanthionine analogs for use in peptide synthesis ................................................... 127 4.4.3 LS can produce meso-lanthionine in the presence of D-cysteine ............................ 128 4.4.4 Availability of D-cysteine ........................................................................................ 128 4.4.5 Fn1732 as a potential lanthionine epimerase .......................................................... 129 4.4.6 Potential inhibitors of LS ........................................................................................ 131 4.5 Concluding Remarks ...................................................................................................... 133 Chapter 5: Lysine 5,6-Aminomutase and KamBC ................................................................ 135 5.1 Chapter Summary .......................................................................................................... 135 5.2 Experimental Procedures ............................................................................................... 136 5.2.1 Materials ................................................................................................................. 136 5.2.2 Cloning and expression of 5,6-LAM, KamBC, and DAHDH ................................ 136 5.2.3 Enzyme purification of 5,6-LAM, KamBC, and DAHDH ..................................... 138 5.2.4 Gel filtration chromatography ................................................................................ 139 5.2.5 Aerobic UV-visible spectroscopy ........................................................................... 140 5.2.6 Anaerobic UV-visible spectroscopy ....................................................................... 140 5.2.7 Steady-state kinetic assays measuring 5,6-LAM turnover ..................................... 141 5.2.8 Steady-state kinetic assays measuring ATPase activity of KamBC ....................... 142 5.2.9 Native-PAGE .......................................................................................................... 143 5.3 Results ............................................................................................................................ 143 5.3.1 Properties of purified 5,6-LAM, KamBC, and DAHDH ........................................ 143 5.3.2 Gel filtration chromatography ................................................................................ 144 5.3.3 UV-visible spectroscopy ......................................................................................... 146 5.3.4 Steady-state kinetic analysis of 5,6-LAM turnover ................................................ 151 5.3.5 Steady-state kinetic analysis of KamBC ATPase activity ...................................... 153 xi 5.3.6 Native-PAGE analysis ............................................................................................ 155 5.4 Discussion ...................................................................................................................... 155 5.4.1 5,6-LAM undergoes suicide inactivation during catalysis ..................................... 155 5.4.2 Reactivation of 5,6-LAM requires KamBC, ATP, and AdoCbl ............................. 156 5.4.3 Metallochaperone systems of AdoCbl-dependent enzymes are structurally distinct..... ................................................................................................................................ 157 5.4.4 KamBC is specific for 5,6-LAM ............................................................................ 160 5.4.5 OraT as a potential reactivating factor for 4,5-OAM ............................................. 161 5.5 Concluding Remarks ...................................................................................................... 162 Chapter 6: Conclusion .............................................................................................................. 164 References .................................................................................................................................. 167 xii List of Tables Table 2.1 Steady-state kinetic parameters associated with the production of H2S by wild-type serine synthase or D232N with increasing amounts of L-cysteine (serine synthase reaction). ................................................................................................................. 41 Table 2.2 Steady-state kinetic parameters associated with the production of L-cysteine by serine synthase in the presence of L-serine and Na2S (cysteine synthase reaction)......... ......................................................................................................... 42 Table 2.3 The rate of pyruvate production for wild-type serine synthase or D232N in the presence of L-cysteine, NADH, and lactate dehydrogenase. .................................. 43 Table 2.4 Pre-steady-state kinetic parameters associated with the reaction between wild-type serine synthase or D232N and L-cysteine. .............................................................. 47 Table 2.5 Pre-steady-state kinetic parameters associated with the reaction between wild-type serine synthase or D232N and L-serine. ................................................................. 51 aTable 3.1 Forward and reverse primers used in PCR to generate different SS variants. ....... 67 Table 3.2 Steady-state kinetic parameters associated with H2S production by SS variants in the presence of lead acetate and L-cysteine. ................................................................. 75 Table 3.3 Steady-state kinetic parameters associated with L-cysteine production by SS variants in the presence of L-serine and Na2S. ..................................................................... 76 Table 3.4 Pre-steady-state kinetic parameters for the reactions of SS variants with L-cysteine. ................................................................................................................................ 79 Table 3.5 Pre-steady-state kinetic parameters for the reactions of SS variants with L-serine. 85 Table 3.6 List of tested nucleophiles and their respective structures. .................................... 93 aTable 4.1 Steady-state kinetic parameters associated with meso-lanthionine or D,L-lanthionine methyl ester production by lanthionine synthase in the presence of OAS and D-cysteine or D-cysteine methyl ester, respectively, and meso-lanthionine production by Fn1732 in the presence of L,L-lanthionine. ...................................................... 112 Table 4.2 Pre-steady-state kinetic parameters for the reactions of lanthionine synthase with OAS and L-cysteine or D-cysteine at 460 nm and 428 nm. .................................. 117 Table 4.3 Absorbance maxima (lmax) determined from the absorbance spectra of lanthionine synthase with potential inhibitors. ........................................................................ 121 xiii Table 4.4 Structures and experimentally determined Kd values for potential inhibitors of LS. . .............................................................................................................................. 123 Table 4.5 Inhibition constants of L,L-DAP and meso-DAP for lanthionine synthase. .......... 125 aTable 5.1 Forward and reverse primers used in PCR to amplify 5,6-LAM, KamBC, and DAHDH. .............................................................................................................. 137 Table 5.2 PCR cycling parameters for amplification of 5,6-LAM, KamBC, and DAHDH. 137 Table 5.3 Quaternary structure determination of 5,6-LAM, KamBC, and DAHDH based on estimated molecular weights by size-exclusion chromatography. ....................... 145 Table 5.4 Anaerobic rates of inactivation of 5,6-LAM with b-L-lysine in different reaction conditions determined from the change in absorbance at 355 nm over time. ...... 149 Table 5.5 Steady-state kinetic parameters associated with the production of erythro-3,5-diaminohexanoate from the reaction of 5,6-LAM with b-L-lysine. ..................... 152 Table 5.6 Rate of ATP hydrolysis with D,L-lysine or β-L-lysine in the presence and absence of KamBC, 5,6-LAM, and AdoCbl. ......................................................................... 154 xiv List of Figures Figure 1.1 The organization of bacteria in the biofilm. ............................................................. 2 Figure 1.2 Mechanisms by which F. nucleatum adhesins affect the progression of colorectal cancer. ...................................................................................................................... 4 Figure 1.3 Enzymes from F. nucleatum involved in L-cysteine-dependent H2S biosynthesis. . 6 Figure 1.4 Conversion of the internal aldimine to the external aldimine through gem-diamine intermediates. ........................................................................................................... 6 Figure 1.5 Diversity of PLP-dependent reactions. ..................................................................... 7 Figure 1.6 Serine synthase reaction. .......................................................................................... 9 Figure 1.7 Serine synthase reaction mechanism. ..................................................................... 10 Figure 1.8 Serine synthase structure and active site. ............................................................... 11 Figure 1.9 Structure and reaction of tryptophan synthase. ...................................................... 13 Figure 1.10 Peptidoglycan layer composition in F. nucleatum. ................................................ 14 Figure 1.11 Lanthionine synthase reaction. ............................................................................... 15 Figure 1.12 Lanthionine synthase reaction mechanism. ............................................................ 16 Figure 1.13 Lanthionine synthase structure and active site. ...................................................... 17 Figure 1.14 CBS and OASS canonical reactions. ...................................................................... 18 Figure 1.15 Lanthionine analog reactions. ................................................................................ 19 Figure 1.16 DAP epimerase reaction mechanism. ..................................................................... 20 Figure 1.17 Lysine fermentation operon and metabolic pathway of L-lysine degradation. ....... 22 Figure 1.18 Structure of the adenosylcobalamin cofactor. ........................................................ 23 Figure 1.19 Characteristic isomerization reaction catalyzed by AdoCbl-dependent isomerases. . ................................................................................................................................ 24 Figure 1.20 5,6-LAM reaction mechanism. ............................................................................... 25 Figure 1.21 The “edge-on” and “top-on” conformations of 5,6-LAM. ..................................... 26 aFigure 2.1 SDS-PAGE gel of purified serine synthase. ........................................................... 36 Figure 2.2 Representation of the enolimine and ketoenamine tautomers of the PLP cofactor. .. ................................................................................................................................ 37 Figure 2.3 Absorbance spectra of serine synthase with L-cysteine and L-serine. .................... 38 Figure 2.4 Peak deconvolution analysis of serine synthase with L-cysteine and L-serine. ...... 39 Figure 2.5 Absorbance spectra of D232N with L-cysteine and L-serine. ................................. 40 xv Figure 2.6 Determination of steady-state kinetic parameters associated with H2S production for wild-type serine synthase and D232N. ................................................................... 41 Figure 2.7 Determination of steady-state kinetic parameters associated with L-cysteine production for serine synthase. ............................................................................... 42 Figure 2.8 Transimination reaction of K46 with the a-aminoacrylate intermediate. .............. 43 Figure 2.9 Rate of pyruvate production for wild-type serine synthase and D232N. ............... 44 Figure 2.10 Stopped-flow spectra of serine synthase with L-cysteine. ...................................... 45 Figure 2.11 Stopped-flow spectra of D232N with L-cysteine. .................................................. 46 Figure 2.12 Rates associated with the formation of the external aldimine or a-aminoacrylate intermediate in the reaction of serine synthase with L-cysteine. ............................ 46 Figure 2.13 Reaction of serine synthase with varying concentrations of L-cysteine. ................ 48 Figure 2.14 Reaction of D232N with varying concentrations of L-cysteine. ............................ 49 Figure 2.15 Stopped-flow spectra of wild-type serine synthase and D232N with L-serine. ...... 50 Figure 2.16 Rates associated with the formation of the external aldimine or a-aminoacrylate intermediate in the reaction of serine synthase with L-serine. ................................ 51 Figure 2.17 Reaction of serine synthase with increasing L-serine concentrations. .................... 52 Figure 2.18 Reaction of D232N with increasing L-serine concentrations. ................................ 53 Figure 2.19 Fluorescence emission spectra of wild-type serine synthase and D232N with L-cysteine and L-serine at 320 nm. ............................................................................ 54 Figure 2.20 Fluorescence emission spectra of wild-type serine synthase and D232N with L-cysteine and L-serine at 295 nm. ............................................................................ 56 Figure 2.21 Superposition of SS and CBS structures. ............................................................... 58 Figure 2.22 Primary sequence alignment of SS with CBS and OASS enzymes from different organisms. .............................................................................................................. 59 Figure 2.23 Predicted pathways for concerted and stepwise mechanisms of serine synthase. . 62 aFigure 3.1 Absorbance spectra of SS variants with L-cysteine and L-serine. .......................... 71 Figure 3.2 Peak analysis of SS variants with L-cysteine and L-serine. .................................... 73 Figure 3.3 Catalytic cycle of the serine synthase reaction with L-cysteine. ............................ 74 Figure 3.4 Determination of steady-state kinetic parameters associated with H2S production for SS variants. ............................................................................................................. 75 xvi Figure 3.5 Determination of steady-state kinetic parameters associated with L-cysteine production for SS variants. ..................................................................................... 77 Figure 3.6 Stopped-flow spectra of SS variants with L-cysteine. ............................................ 78 Figure 3.7 Reaction of W99A with increasing concentrations of L-cysteine. ......................... 80 Figure 3.8 Reaction of F125A with increasing concentrations of L-cysteine. ......................... 81 Figure 3.9 Reaction of F148A with increasing concentrations of L-cysteine. ......................... 82 Figure 3.10 Reaction of F234A with increasing concentrations of L-cysteine. ......................... 83 Figure 3.11 Stopped-flow spectra of SS variants and L-serine. ................................................. 84 Figure 3.12 Reaction of W99A with increasing concentrations of L-serine. ............................. 86 Figure 3.13 Reaction of F125A with increasing concentrations of L-serine. ............................ 87 Figure 3.14 Reaction of F148A with increasing concentrations of L-serine. ............................ 88 Figure 3.15 Reaction of F234A with increasing concentrations of L-serine. ............................ 89 Figure 3.16 Fluorescence emission spectra of SS variants with L-cysteine and L-serine at 320 nm. ................................................................................................................................ 91 Figure 3.17 Fluorescence emission spectra of SS variants with L-cysteine and L-serine at 295 nm. ................................................................................................................................ 92 Figure 3.18 Reaction of a nucleophile with the a-aminoacrylate intermediate to form a new b-substituted amino acid. ........................................................................................... 93 Figure 3.19 Absorbance spectra of SS variants with different nucleophiles. ............................ 95 Figure 3.20 Absorbance spectra of F125A/F148A with L-serine. ............................................. 95 Figure 3.21 Anion-aromatic interactions in KSI and SS. .......................................................... 98 aFigure 4.1 SDS-PAGE gel of purified Fn1732. ..................................................................... 110 Figure 4.2 Absorbance spectra of wild-type lanthionine synthase and S224A with OAS and cysteine analogs. ................................................................................................... 111 Figure 4.3 Determination of steady-state kinetic parameters associated with lanthionine production for lanthionine synthase and Fn1732. ................................................ 113 Figure 4.4 HPLC analysis of lanthionine analogs produced by lanthionine synthase. .......... 114 Figure 4.5 Lanthionine synthase reaction of L-cysteine and D-cysteine to form L,L-lanthionine, meso-lanthionine, and H2S. .................................................................................. 114 Figure 4.6 HPLC analysis of L,L-lanthionine and meso-lanthionine production by lanthionine synthase. ............................................................................................................... 115 xvii Figure 4.7 Stopped-flow spectra of lanthionine synthase with OAS and L-cysteine or D-cysteine. ................................................................................................................ 116 Figure 4.8 Intermediates involved in the reaction of cysteine with the a-aminoacrylate intermediate. ......................................................................................................... 117 Figure 4.9 Reaction of lanthionine synthase with OAS and increasing concentrations of L-cysteine. ................................................................................................................ 119 Figure 4.10 Reaction of lanthionine synthase with OAS and increasing concentrations of D-cysteine. ................................................................................................................ 120 Figure 4.11 Absorbance spectra of lanthionine synthase with potential inhibitors. ................ 122 Figure 4.12 Fluorescence emission spectra of lanthionine synthase with potential inhibitors.124 Figure 4.13 Inhibition of lanthionine synthase by L,L-DAP and meso-DAP. .......................... 125 Figure 4.14 Molecular model of DAP analogs in lanthionine synthase. ................................. 126 Figure 4.15 Sequence alignment of Fn1732 with other PLP-independent epimerases/racemases. .............................................................................................................................. 130 Figure 4.16 Hydrogen bond contacts of lanthionine synthase within the L,L-lanthionine external aldimine. ............................................................................................................... 132 Figure 4.17 Molecular model of meso-oxa-DAP in the active site of lanthionine synthase. .. 133 aFigure 5.1 SDS-PAGE gel of 5,6-LAM, KamBC, and DAHDH. ......................................... 144 Figure 5.2 Estimated molecular weights of 5,6-LAM, KamBC, and DAHDH from gel filtration chromatography. ................................................................................................... 145 Figure 5.3 Estimated hydrodynamic radius of 5,6-LAM, KamBC, and DAHDH. ............... 146 Figure 5.4 PLP and AdoCbl absorbance spectra of 5,6-LAM. .............................................. 147 Figure 5.5 Aerobic absorbance spectra of 5,6-LAM with b-L-lysine in the absence and presence of KamBC alone and with ADP or ATP. ............................................................. 148 Figure 5.6 Anaerobic absorbance spectra of 5,6-LAM with b-L-lysine in the absence and presence of KamBC with AMPPNP, ADP, or ATP. ........................................... 150 Figure 5.7 Aerobic absorbance spectra of 4,5-OAM with D-ornithine in the absence and presence of KamBC and ATP. ............................................................................. 151 Figure 5.8 Determination of steady-state kinetic parameters associated with erythro-3,5-diaminohexanoate production for 5,6-LAM. ....................................................... 152 Figure 5.9 KamBC and ATP-dependent reactivation of inactive 5,6-LAM. ......................... 153 xviii Figure 5.10 Rate of ATP hydrolysis for KamBC in varying conditions. ................................ 154 Figure 5.11 Native-PAGE gel of 5,6-LAM and KamBC. ....................................................... 155 Figure 5.12 Structural comparison of MutS with KamC. ........................................................ 158 Figure 5.13 Structure of diol dehydratase reactivase and diol dehydratase. ........................... 159 Figure 5.14 Structural comparison of 5,6-LAM and 4,5-OAM. ............................................. 160 Figure 5.15 Ornithine fermentation operon and metabolic pathway of L-ornithine degradation... .............................................................................................................................. 162 xix List of Abbreviations 2,3-LAM lysine 2,3-aminomutase 2YT 2x yeast extract tryptone medium 4,5-OAM ornithine 4,5-aminomutase 5,6-LAM lysine 5,6-aminomutase Ado-CH2· 5′-deoxyadenosyl radical Ado-CH3 5′-deoxyadenosine AdoCbl adenosylcobalamin ADP adenosine 5′-diphosphate AKPT 2-amino-4-ketopentanoate thiolase AMPPNP adenylyl-imidodiphosphate ATCC American Tissue Culture Collection ATP adenosine 5′-triphosphate CBS cystathionine-b-synthase cDNA complementary deoxyribonucleic acid CoA coenzyme A CV column volumes Da Dalton DADH 2,4-diaminopentanoate dehydrogenase DAHDH erythro-3,5-diaminohexanoate dehydrogenase DAP 2,6-diaminopimelic acid DAPDH diaminopimelic acid dehydrogenase DMB 5,6-dimethylbenzimidazole DNA deoxyribonucleic acid xx dNTP deoxynucleotide triphosphate DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EPPS 4-(2-hydroxyehtyl)-1-piperazinepropanesulfonic acid EPR electron paramagnetic resonance FRET Forster resonance energy transfer G3P glyceraldehyde-3-phosphate Gal-GalNAc galactose-N-acetylgalactosamine HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC high performance liquid chromatography IGP indole-3-glycerol phosphate IPTG isopropyl b-D-1-thiogalactopyranoside ITC isothermal titration calorimetry KSI ketosteroid isomerase LB Luria-Bertani broth LS lanthionine synthase MW molecular weight MWCO molecular weight cut-off NAD+ nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide (reduced) NADP+ nicotinamide adenine dinucleotide phosphate NADPH nicotinamide adenine dinucleotide phosphate (reduced) NAG N-acetylglucosamine NAM N-acetylmuramic acid xxi ncAA noncanonical amino acid Ni-NTA Ni2+-nitrilotriacetic acid NMR nuclear magnetic resonance NOD nucleotide binding oligomerization domain protein OAS O-acetyl-L-serine OASS O-acetylserine sulfhydrylase OD optical density PAGE polyacrylamide gel electrophoresis PAMP pathogen associated molecular pattern PCR polymerase chain reaction PDB Protein Data Bank PG peptidoglycan PLP pyridoxal 5′-phosphate ROS reactive oxygen species SDS sodium dodecyl sulfate SS serine synthase TB Terrific broth TIM triosephosphate isomerase Tris tris(hydroxymethyl)aminomethane TrpS tryptophan synthase UV ultraviolet xxii Acknowledgements I would like to thank my supervisor, Dr. Kirsten Wolthers, for her continuing guidance and support throughout my degree. The knowledge and experience you have supplied me with is invaluable. I thank the other members of my committee, Dr. Fred Menard and Dr. Thuy Dang, for their advice and helpful discussions. I extend thanks to Dr. Rob Mothersole for being a mentor to me during the first year of my studies. Thank you to the other members of my lab group for their friendship and always providing a positive lab environment (lab rats forever!). To my grandparents, parents, siblings, and the rest of my family and friends, thank you for always supporting me. Finally, I would like to thank my best friend and partner, Anthony. I am grateful for your endless encouragement, patience, and assistance inside and outside of the lab. I am looking forward to whatever comes next for us. xxiii For my cat, Nellie, who has remained a constant source of unconditional love and comfort. This thesis would have been completed sooner had she stopped sitting on the keyboard. 1 Chapter 1: Introduction 1.1 Fusobacterium nucleatum 1.1.1 Fusobacterium nucleatum as an opportunistic pathogen Despite the broad range of bacterial diversity, microbiological research has prioritized certain phyla, such as Proteobacteria, due to their significance in human health and agriculture, unique metabolic capabilities, and relative ease of study.1 However, this focus is limiting as there are a wide variety of unexplored bacteria that impact human health and the environment. A prime example of understudied microorganisms is those that belong to Fusobacteria, a distinct phylum of bacteria. Fusobacterium species within this phylum are often found in the mouth or other mucosal sites within humans and animals. Out of the species found in the human oral cavity, Fusobacterium nucleatum is the most abundant.2 While it is a natural constituent of the oral microbiome, its frequent isolation and identification in patients with different infections has resulted in F. nucleatum being regarded as an opportunistic pathogen.3 Thus, this bacterium has emerged at the forefront of scientific research due to an increasing number of associations with oral and extra-oral diseases. 1.1.2 F. nucleatum in the oral cavity In the oral cavity, the gram-negative anaerobe Fusobacterium nucleatum participates as a bridging organism by facilitating interactions between early colonizers and late colonizers of the dental plaque.4 Early colonizers are primarily composed of gram-positive species such as Actinomyces and Streptococcus, and late colonizers are predominantly gram-negative species that include Treponema denticola and Porphyromonas gingivalis.5 The formation of the biofilm environment is initiated through the adherence of early colonizers to the tooth’s surface, followed by late colonizers that rely on interbacterial adhesion to facilitate their entry into the plaque community.6 F. nucleatum mediates this biofilm organizing behaviour through the expression of adhesion proteins such as Aid1, RadD, and CmpA that bind to early colonizers, and FomA and Fap2 that bind to late colonizers (Figure 1.1).4,7–10 The adhesive properties of F. nucleatum allow the formation of a symbiotic relationship with other bacteria, but the ability to bind to human cells results in a relationship that can be pathogenic. 2 Figure 1.1 The organization of bacteria in the biofilm. Early colonizers such as Streptococcus spp. and Actinomyces spp. adhere to the tooth surface. F. nucleatum can bind to the early colonizers with adhesion proteins Aid1, CmpA, and RadD. Using adhesins FomA and Fap2, F. nucleatum can also adhere to late colonizers Treponema denticola and Porphyromonas gingivalis. These interspecies interactions facilitated by F. nucleatum contribute to the organization of bacteria in the formation of the biofilm. F. nucleatum occupies the oral cavity in healthy individuals, but it also plays a role in the development of periodontal disease. Using its adhesion proteins, F. nucleatum can bind to and invade gingival epithelial cells, disrupting of the connection between cells.11 Disruption of the host epithelium prompts increased infectivity and invasion potential of other periodontal pathogens such as P. gingivalis.12 Working together, these bacteria can evade the host immune system and form an environment that promotes inflammation and disease progression. The host defends against bacterial colonization through the secretion of the antimicrobial peptide b-defensin.13 Recognition of F. nucleatum also results in induced expression of pro-inflammatory cytokines IL-1b, IL-6, and TNF-a.14 This inflammatory response can lead to tissue destruction and bone loss that is characteristic of periodontal disease. Amino acid metabolism is another route for F. nucleatum to exert its virulence factors. F. nucleatum preferentially obtains its energy through the fermentation of amino acids such as ToothStreptococcus spp.Actinomyces spp.Fusobacterium nucleatumTreponema denticolaPorphyromonas gingivalisAid1CmpARadDFomAFap2Biofilm 3 glutamate, histidine, lysine, and serine.15 A major product of fermentation, butyrate, facilitates the advancement of periodontal disease as it can inhibit proliferation of gingival fibroblasts, causing rapid wound healing to be compromised.16,17 Another metabolite of F. nucleatum that contributes to the progression of periodontal disease is hydrogen sulfide (H2S). Hydrogen sulfide is a toxic, volatile sulfur compound that is formed through the degradation of sulfur-containing amino acids such as L-cysteine. When present in the oral cavity, increased levels of H2S can damage epithelial cells, enhance the permeability of the oral membrane, and induce apoptosis of gingival fibroblasts.18–20 1.1.3 Associations to extra-oral diseases The adhesive and invasive properties of F. nucleatum also allow it to disseminate into the bloodstream and colonize extra-oral sites, which is associated to different pathologies. Examples of F. nucleatum-associated diseases include adverse pregnancy outcomes and colorectal cancer. These conditions are often thought to have an oral source of infection as F. nucleatum has been detected in the amniotic fluid of pre-term births and colorectal cancer tissue in conjunction with other oral bacteria.21,22 The ability of F. nucleatum to colonize the placenta is through cellular invasion by adhesins Fap2 and FadA.10,23 This was demonstrated in a study with mice where the intravenous injection of F. nucleatum allowed it to adhere to and invade endothelial cells, cross the endothelium, proliferate in surrounding tissues, and spread to the amniotic fluid, resulting in premature delivery, stillbirths, and non-sustained live births.24 F. nucleatum, as an active invader of host cells, is also able to traverse to colorectal cancer tissue from the digestive tract or the bloodstream where it affects multiple stages of colorectal cancer and shapes the tumor microenvironment. In the initial stage of tumorigenesis, the proliferation of cancer cells is promoted through the binding of fusobacterial adhesin FadA to the tumor suppressor E-cadherin on the surface of colonic epithelial cells (Figure 1.2). This inhibitory interaction results in the activation of b-catenin and Wnt signaling, leading to increased expression of transcription factors, oncogenes, inflammatory genes, and growth stimulation of colorectal cancer cells.25 In the process of tumor development, F. nucleatum can promote a pro-inflammatory environment and suppress the host immune response.26 Regulation of the immune response can be achieved through 4 F. nucleatum amino acid fermentation; the production of butyrate recruits myeloid-derived suppressor cells that inhibit T cell proliferation and induce T cell apoptosis.27 Fap2 on the surface of F. nucleatum can also suppress T cell activation and the killing of tumor cells by binding to an immune inhibitory receptor on the cell surface.28 If a tumor develops, Fap2 can also bind to galactose-N-acetylgalactosamine (Gal-GalNAc), a sugar residue that is overexpressed on the surface of colorectal cancer cells.29 Production of H2S by F. nucleatum also plays a role in the pathogenesis of colorectal cancer by promoting angiogenesis, which consequently supports tumor growth and metastasis.30,31 The enrichment of F. nucleatum in colorectal cancer tissue via these mechanisms results in associations with poor patient prognosis as well as cancer recurrence, as F. nucleatum is also able to promote chemoresistance by modulating autophagy.32,33 Figure 1.2 Mechanisms by which F. nucleatum adhesins affect the progression of colorectal cancer. The fusobacterial adhesin Fap2 can bind to an immune inhibitory receptor on the surface of T cells where it inhibits T cell activation and the killing of tumor cells. Enrichment of F. nucleatum in colorectal cancer cells results from Fap2 binding to the overexpressed sugar Gal-GalNAc on the cell surface. Cell proliferation is initiated by the adhesive protein FadA binding to E-cadherin on the surface of colonic epithelial cells. This results in the activation of the catenin and Wnt pathways that are often dysregulated during cancer. Fap2immune inhibitory receptorkilling tumor cellsT cellGal-GalNAcFap2colorectal cancer cellFadAE-cadherinβ-catenin and Wntcolonic epithelial cellcell proliferationF. nucleatum 5 1.1.4 H2S production by F. nucleatum In humans, H2S serves as a biological mediator by regulating physiological processes in the nervous, cardiovascular, immune, and gastrointestinal systems.34 At low concentrations, H2S can have beneficial antioxidant/cytoprotective effects as well as regulation of vascular tone and blood pressure, stimulation of cellular respiration, and suppressed inflammation.35–37 However, elevated levels of H2S are associated with various respiratory infections such as chronic bronchitis, emphysema, and pneumonia, as well as cardiovascular-related diseases such as hypertension.36 The progression of other disease conditions, such as colorectal cancer, has been linked to increased levels of H2S due to the up-regulation of H2S-producing enzymes.30 In mammalian cells, three enzymes within the cysteine biosynthesis pathway are involved in H2S production: cystathionine-b-synthase, cystathionine-g-lyase, and 3-mercaptopyruvate sulfurtransferase.38 Analysis of bacterial genomes revealed that most bacteria encode homologs of these enzymes with the same function, suggesting an important physiological function of H2S.39 The exact biological role of H2S in bacteria is unknown, however, it has been suggested to make bacteria more resistant to the host immune response and oxidative stress induced by some antibiotics.34,39,40 F. nucleatum, a prominent member of the oral microbiome, serves as the primary source of H2S within the oral cavity. The F. nucleatum genome has four genes (Fn0625, Fn1055, Fn1220, and Fn1419) that encode enzymes that are involved in H2S production via L-cysteine metabolism (Figure 1.3). Fn0625 and Fn1419 encode for a L-cysteine desulfhydrase and methionine-g-lyase, respectively. These enzymes catalyze the a,b-elimination reaction of L-cysteine and H2O to form H2S, pyruvate, and ammonia.41,42 Methionine-g-lyase can also catalyze the a,g-elimination of L-methionine to produce methyl mercaptan, a-ketobutyrate, and ammonia.42 Fn1055 encodes for a cysteine (hydroxyl) lyase or serine synthase (hereafter identified by the latter name) and catalyzes the b-replacement reaction of L-cysteine and H2O to form L-serine and H2S.43 Fn1220 has been characterized as a lanthionine synthase, catalyzing the condensation of two equivalents of L-cysteine to produce L,L-lanthionine and H2S.44 These H2S-producing enzymes, along with other enzymes involved in amino acid metabolism, are dependent on the vitamin B6 cofactor, pyridoxal 5′-phosphate (PLP). 6 Figure 1.3 Enzymes from F. nucleatum involved in L-cysteine-dependent H2S biosynthesis. 1.1.5 Pyridoxal 5′-phosphate-dependent enzymes Pyridoxal 5′-phosphate (PLP)-dependent enzymes have the PLP cofactor covalently bound to the enzyme through an imine linkage that is formed in the condensation reaction of the PLP aldehyde group and the terminal amine of an active site lysine residue. This forms a Schiff base, also known as the internal aldimine. The binding of an amino acid substrate results in the displacement of the lysine residue to form the external aldimine. This intermediate is preceded by a gem-diamine intermediate in which both the lysine residue and the substrate are bound to the C4′ of the PLP cofactor (Figure 1.4). Figure 1.4 Conversion of the internal aldimine to the external aldimine through gem-diamine intermediates. L-cysteine+ H2SHSCOO-NH2L,L-lanthionineSCOO-NH2-OOCNH2L-serineHOCOO-NH2Fn1055H2OL-cysteineHSCOO-NH2+ H2SFn1220H2ONH3pyruvateO-OOH2S +Fn0625Fn1419NO-2-O3PONH+Lysinternal aldimineNO-2-O3PONHLysgem-diamine IR COO-NH2+H2N-OOC RNH2LysNO-2-O3PONH+external aldimineCOO-RNO-2-O3PONH2+Lysgem-diamine IIHN-OOC R 7 The formation of the external aldimine is a common first catalytic step of PLP-dependent enzymes. The next step, however, is where the most variability lies in the reaction mechanism and is largely dictated by the noncovalent interactions between active site residues and the chemical groups of the external aldimine.45 Heterolytic cleavage occurs at a bond to the Ca (Ca-H, Ca-CO2, or Ca-R) to generate a carbanion intermediate. The stabilization of the Ca-anion is facilitated by the delocalization of the negative charge through the pyridine ring of the PLP cofactor, thus allowing PLP to function as an electron sink in most PLP-dependent enzymes.46 The variability in which bond is broken at the Ca results in PLP-dependent enzymes being able to catalyze a diverse number of reactions including racemization, transamination, decarboxylation, and elimination or replacement at Cb or Cg (Figure 1.5). Figure 1.5 Diversity of PLP-dependent reactions. The mechanism of PLP-dependent enzyme reactions diverges through which bond to the Ca is broken in the external aldimine. Removal of the side chain (R) results in a side chain cleavage reaction (magenta arrow), proton abstraction results in racemization, transamination, or b- or g-elimination/replacement reactions (red arrow), and removal of the carboxylate results in a decarboxylation reaction (blue arrow). NO-2-O3PONH+external aldimineCOO-RHNO-2-O3PONH+COO-RNO-2-O3PONH+HRH+CO2racemization,transamination,β- or 훾-elimination/replacementdecarboxylationNO-2-O3PONH+COO-Hside chain cleavageR+NOH2-O3PONLysinternal aldimineR COO-NH2 8 1.1.6 Fold types of PLP-dependent enzymes PLP-dependent enzymes are classified by their three-dimensional folds and can be divided into seven fold types.47,48 Notably, each fold type can catalyze multiple types of reactions. The most structurally and functionally diverse family of PLP-dependent enzymes is the fold type I enzymes, which include aminotransferases, decarboxylases, and some enzymes with a-, b-, or g-elimination activity.49 Fold type I enzymes usually exist as homodimers or homotetramers, with each subunit containing a large N-terminal domain and a small C-terminal domain. Each subunit contains a PLP cofactor, which is bound by a lysine residue near the C-terminus. Preceding the lysine residue is a conserved aspartic acid residue that interacts with the pyridine nitrogen of PLP.50 A glycine-rich loop near the N-terminus is responsible for binding the PLP phosphate group. The active site is located at the interface between the subunits, with both subunits and both domains contributing residues for catalysis.51 In contrast to fold type I enzymes, the residues involved in catalysis in fold type II enzymes originate from one subunit. These enzymes are functional as dimers, tetramers, and oligomers, and contain two domains that consist of a mixed a/b structure. The PLP cofactor is bound between these two domains, forming a Schiff base between the PLP cofactor and a lysine residue at the N-terminus. The PLP phosphate group binds to the loop region near the C-terminus and the PLP pyridine nitrogen coordinates to a serine residue that is in the equivalent position as the aspartic residue in fold type I enzymes.45 Examples of PLP-dependent enzymes that belong to the fold type II family include tryptophan synthase, O-acetylserine sulfhydrylase, cystathionine b-synthase, and threonine deaminase. The fold type III enzymes include alanine racemase and eukaryotic ornithine decarboxylase. These enzymes have a similar arrangement of residues that interact with the PLP cofactor compared to fold type II enzymes (a lysine residue at the N-terminus and a glycine loop at the C-terminus).45 In alanine racemase, the residue forming a hydrogen bond with the PLP pyridine nitrogen is an arginine residue, which is different from both fold type I and type II enzymes.51 However, ornithine decarboxylase has a similar environment around the pyridine nitrogen as fold type I enzymes, with a hydrogen bond contact to an acidic glutamic acid residue.52 This interaction is also formed in fold type IV enzymes, which include D-alanine aminotransferase and branched chain aminotransferase.51 In the fold type V enzymes, there are no hydrogen bonds between active site residues and the pyridine nitrogen of the PLP cofactor. These enzymes, including glycogen phosphorylase, differ from the other fold types in that the PLP cofactor does not function as an electrophilic catalyst. Instead, its phosphate group participates in 9 proton transfer during catalytic turnover.51 PLP also has a different role in fold type VI and VII enzymes lysine 5,6-aminomutase (5,6-LAM) and lysine 2,3-aminomutase (2,3-LAM), respectively. These enzymes utilize an additional cofactor (adenosylcobalamin in 5,6-LAM; S-adenosylmethionine in 2,3-LAM) to facilitate difficult 1,2-rearrangement reactions through radical catalysis. The non-traditional role of PLP in these reactions is to lower the energy barrier for intramolecular isomerization by introducing unsaturation into the migrating amino group.53 1.2 Serine synthase 1.2.1 Serine biosynthesis Serine synthase (SS; Fn1055) is a fold type II PLP-dependent enzyme that produces L-serine and H2S in F. nucleatum through the b-replacement reaction of L-cysteine and H2O (Figure 1.6). Serine is important in metabolism as it is a precursor for the biosynthesis of amino acids, proteins, and phospholipids.54 In most bacteria, the biosynthesis of L-serine from 3-phosphoglycerate comprises three sequential enzymatic steps catalyzed by 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase. This complete phosphorylated pathway is absent from the F. nucleatum genome. Moreover, F. nucleatum does not encode a homolog of serine hydroxymethyltransferase, the enzyme that interconverts glycine and L-serine.55 Thus, serine synthase may play a pivotal role in maintaining L-serine homeostasis in F. nucleatum. Figure 1.6 Serine synthase reaction. 1.2.2 Serine synthase reaction mechanism The resting form of serine synthase contains the PLP cofactor that is bound to the enzyme through an imine linkage with K46 to form the internal aldimine (Figure 1.7). The binding of L-cysteine leads to the formation of the external aldimine. Proton abstraction from the Ca by the terminal amino group of K46 leads to a Ca carbanion that is resonance stabilized to form a quinonoid COO-HSNH2H2O+COO-HONH2H2S+L-cysteine L-serineSS 10 intermediate. The a-aminoacrylate intermediate is formed following the b-elimination of the thiol group from L-cysteine. An active site aspartic acid residue, D232, acts as a general base catalyst to activate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. This results in the formation of a second carbanionic/quinonoid species. Re-protonation of the Ca by K46 generates the external aldimine with L-serine. A transimination reaction occurs between the side chain of K46 and the PLP C4′ of the L-serine external aldimine, resulting in the release of L-serine and the formation of the internal aldimine, completing the catalytic cycle. Figure 1.7 Serine synthase reaction mechanism. 1.2.3 The hydrophobic active site of serine synthase Serine synthase forms a dimer with each subunit comprised of a small N-terminal domain and a large C-terminal domain.56 The N-terminal domain folds into a three-stranded parallel b-sheet that is sandwiched by four a-helices. The C-terminal domain comprises a central seven-stranded mixed NO-2-O3PONH+Lys46NH2Lys46internal aldimineHSCOO-NH2NO-2-O3PONH+external aldimineCOO-HSHNH3+Lys46N-O-2-O3PONH+quinonoidCOO-HSNH3+Lys46NO-2-O3PONH+α-aminoacrylateCOO-H2SOHHNH2Lys46NO-2-O3PONH+external aldimineCOO-HOHNH2+Lys46N-O-2-O3PONH+quinonoidCOO-HOAsp232OHOHHOCOO-NH2Asp232O-OAsp232OHOAsp232OHOAsp232OHOAsp232OHOL-cysteineL-serine 11 b-sheet surrounded by seven a-helices. The structure of serine synthase is homologous to other fold type II PLP-dependent enzymes, except that it has a C-terminal extension that is suggested to contribute to the structural stability and dimerization of the protein.56 At the interface between the two domains is the PLP cofactor, which is bound to the enzyme through a covalent linkage with the e-amino group of K46. As shown in Figure 1.8, the PLP cofactor in the open conformation is exposed to the solvent through a narrow tunnel of hydrophobic residues (W99, F125, F148, and F234). Substrate binding triggers closure of the active site, and these aromatic residues undergo a conformational change to shield the PLP cofactor from the solvent. The presence of these residues surrounding D232 also likely increases the basicity of the carboxylate side chain in order for it to act as a general base during catalysis. Figure 1.8 Serine synthase structure and active site. A superposition of the open conformation in chain B (grey) and the closed conformation in chain A (cyan) of the internal aldimine structure of serine synthase (PDB entry 5Z5C at 2.07 Å resolution).56 A close-up of the active site shows the repositioning of hydrophobic residues W99, F125, F148, and F234. Also depicted is K46 bound to the PLP cofactor in the resting state of the enzyme, and D232, the catalytic acid/base. The PLP cofactor is shown as sticks with the carbon atoms in green and a catalytic water molecule is shown as a red sphere. A hydrogen bond between D232 and H2O in the closed conformation is shown as a black dashed line. D232 W99 F125 F234 F148 K46 12 1.2.4 Noncanonical amino acid synthesis The presence of these aromatic side chains in the active site of serine synthase restricts the number of substrates that can react with it and thus provides a barrier for this enzyme to be used in noncanonical amino acid (ncAA) synthesis. Noncanonical amino acids are any amino acids that are not naturally incorporated into a protein sequence during translation. Although they are often referred to as unnatural amino acids, many ncAAs occur naturally as post-translationally modified peptide residues or as secondary metabolite intermediates.57 Either found in nature or created synthetically, ncAAs are valuable and they can be used as biophysical probes or incorporated into bioactive small molecules and peptide therapeutics.58–60 Unnatural amino acids kynurenine and 3-methyl glutamic acid are incorporated into the synthesis of daptomycin, a drug used to treat gram-positive bacterial infections.59 ncAAs can also be substituted for canonical amino acids in protein synthesis through genetic code expansion. This widely used methodology allows for precise biochemical manipulation of proteins with enhanced or different functionalities.61,62 For example, the replacement of histidine with Nd-methylhistidine as the proximal heme ligand in ascorbate peroxidase and myoglobin results in improved catalytic performance.63,64 Despite its broad range of applications, the chemical synthesis of ncAAs is challenging due to the requirements of precise configuration at the Ca stereocenter and protection of the reactive amino and carboxylate groups. An alternative approach to ncAA synthesis is to use enzymes, which can overcome these stereo- and regio-selective barriers by binding and positioning substrates in the preferred position to generate specific enantiomers. Moreover, enzymes avoid the need for protecting groups. Tryptophan synthase, a fold type II PLP-dependent enzyme, has been successfully engineered for ncAA synthesis and thus serves as a model system. Tryptophan synthase (TrpS) is an a2b2 heterodimer that catalyzes the final steps in the L-tryptophan biosynthetic pathway: the reaction of indole-3-glycerol phosphate (IGP) and L-serine to form glyceraldehyde-3-phosphate (G3P) and L-tryptophan (Figure 1.9).65,66 The mechanism for this enzyme proceeds through allosteric activation of both the a- and b-subunits, deemed TrpA and TrpB, respectively. IGP binding to TrpA initiates a conformational change to activate TrpB for binding L-serine to the PLP cofactor.67 Subsequent b-elimination of the hydroxyl group from L-serine results in the formation of the a-aminoacrylate intermediate, which then activates TrpA to 13 form indole and G3P through the retro-aldol cleavage of IGP.68 Indole gets released into a hydrophobic intramolecular tunnel that extends to TrpB, where it can add to the a-aminoacrylate intermediate to form L-tryptophan.69 Although the a-subunit is important for catalysis, it is unnecessary for the synthesis of ncAAs due to the reaction between indole and the a-aminoacrylate intermediate occurring in the b-subunit. However, the removal of TrpA from the enzyme complex results in a significant decrease in the activity of TrpB.70,71 Directed evolution of TrpB was used to restore its catalytic efficiency to the point of surpassing the activity of the native complex.66 This engineered TrpB activation allowed for a dramatic expansion of the substrate scope for this stand-alone enzyme to form new C-C, C-N, and C-S bonds, producing entirely new-to-nature ncAAs including b-methyltryptophan, dihydroisotryptophan, and S-benzyl-L-cysteine.72–74 Figure 1.9 Structure and reaction of tryptophan synthase. The structure of TrpS from Pyrococcus furiosus (PDB entry 5E0K at 2.76 Å resolution) shows the a- and b-subunits in red and blue, respectively.66 The PLP cofactor is shown as green sticks and was inserted into the structure through a superposition with the structure of TrpB complexed with the L-serine external aldimine (PDB entry 5DW0 at 2.01 Å resolution).66 OPO-OO-OHOHHNOPO-OO-OHOHNHHNCOO-NH2HOCOO-NH2indoleindole-3-glycerol phosphateglyceraldehyde-3-phosphateL-serineL-tryptophanb-subunit (TrpB) a-subunit (TrpA) 14 1.3 Lanthionine synthase 1.3.1 The peptidoglycan layer in F. nucleatum incorporates meso-lanthionine Peptidoglycan (PG) is a structurally integral component of the bacterial cell wall that maintains cellular shape and rigidity, provides a protective barrier, and prevents cell lysis due to osmotic stress.75 The PG layer is composed of repeating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) subunits that are cross-linked via peptide side chains. The overall PG structure is similar across different types of bacteria; however, there are modifications in the peptide crosslink that create diversity between bacterial species. In most bacteria, the first two amino acids in the peptide stem are usually L-alanine and either D-isoglutamate (gram-negative) or D-isoglutamine (gram-positive). All bacteria contain D-alanine in the fourth position. The third position of the peptide crosslink is where the most variation occurs amongst different species of bacteria. Gram-positive bacteria usually incorporate L-lysine in this position. In gram-negative bacteria, the third position is usually occupied by the diamino acid meso-2,6-diaminopimelic acid (meso-DAP). The production of meso-DAP in bacteria occurs at the eighth step of the biosynthetic pathway leading from L-aspartate to L-lysine.76 Interestingly, F. nucleatum lacks this biosynthetic pathway and instead incorporates the thioether analog meso-lanthionine into its peptidoglycan layer (Figure 1.10).77 Figure 1.10 Peptidoglycan layer composition in F. nucleatum. L-alanineD-isoglutamatemeso-lanthionineD-alanineNAMNAMNAGNAGouter membraneinner membranepeptidoglycan layer 15 In F. nucleatum, the only known synthetic route to produce lanthionine is through lanthionine synthase (LS; Fn1220). This enzyme catalyzes the PLP-dependent reaction of two equivalents of L-cysteine to form L,L-lanthionine and H2S (Figure 1.11). Disruption of the gene encoding for lanthionine synthase demonstrated that this enzyme is essential for F. nucleatum viability. Thus, it may serve as a potential drug target for controlling the growth of F. nucleatum.78 Figure 1.11 Lanthionine synthase reaction. 1.3.2 Lanthionine synthase reaction mechanism In the active site of lanthionine synthase, K42 forms an imine linkage with the PLP cofactor to form the internal aldimine. The catalytic cycle begins with the binding of L-cysteine into the active site to generate the external aldimine. Abstraction of the Ca proton by K42 leads to the b-elimination of the thiol group of L-cysteine, releasing H2S and forming the a-aminoacrylate intermediate. A second L-cysteine molecule binds into the second substrate binding pocket and performs a nucleophilic attack on the a-aminoacrylate intermediate. Proton donation by K42 onto the Ca results in the formation of the external aldimine with the product, L,L-lanthionine. Displacement of L,L-lanthionine by the active site lysine residue results in the release of the product and the regeneration of the internal aldimine. The entire reaction mechanism is shown in Figure 1.12. HSCOO-NH2HSNH2+ SCOO-NH2-OOCNH2L-cysteine L-cysteine L,L-lanthionineCOO-+ H2SLS 16 Figure 1.12 Lanthionine synthase reaction mechanism. 1.3.3 Lanthionine synthase structure The structure of lanthionine synthase is similar to other fold type II PLP-dependent enzymes such as cystathionine-b-synthase (CBS), O-acetylserine sulfhydrylase (OASS), and threonine deaminase.79–81 LS forms a dimer, with each monomer comprised of a small N-terminal domain and a large C-terminal domain.82 Both domains are a/b-type and contain central b-sheets surrounded by a-helices. The PLP cofactor is located at the core of the protein at the interface between the two subdomains. The internal aldimine structure is formed through a covalent linkage between the PLP cofactor and the e-amino group of K42. Upon substrate binding, the conserved loop (69TSGNT73) undergoes a conformational shift, triggering closure of the active site (Figure 1.13). Movement of this loop is communicated to the 223MSIG226 loop, which plays an important role in product binding due to the hydrogen bonding interactions between the backbone NH and b-hydroxyl group of S224 to the terminal carboxylate of L,L-lanthionine. Notably, 224SI225 is a NO-2-O3PONH+Lys42NH2Lys42internal aldimineHSCOO-NH2NO-2-O3PONH+external aldimineCOO-HSHNO-2-O3PONH+α-aminoacrylateCOO-H2SNH2Lys42NO-2-O3PONH+external aldimineCOO-SHSCOO-NH2-OOCNH2NH2-OOCS--OOCNH2NH2+Lys42HL-cysteineL-cysteineL,L-lanthionine 17 unique insertion element of LS that is not present in other fold type II PLP-dependent enzymes.44 This may account for the evolution of this enzyme towards lanthionine biosynthesis. Figure 1.13 Lanthionine synthase structure and active site. The open conformation of LS in the internal aldimine form (grey; PDB entry 5XEO at 2.03 Å resolution) is overlayed with the closed conformation of LS complexed with L,L-lanthionine (violet; PDB entry 5XEM at 1.84 Å resolution).82 A close-up of the active site shows the reorganization of the amino acid residues within the 69TSGNT73 and 223MSIG226 loops. The carbons of the PLP cofactor are green and the carbons of lanthionyl are cyan. Only the side chains of active site residues T69, S70, S224 and I225 are shown for clarity. 1.3.4 Fold type II PLP-dependent enzymes exhibit substrate promiscuity Cystathionine-b-synthase (CBS) and O-acetylserine sulfhydrylase (OASS) catalyze b-replacement reactions through an a-aminoacrylate intermediate. The canonical reaction of CBS is the condensation reaction of L-serine and L-homocysteine to form L-cystathionine and H2O (Figure 1.14). This reaction is the first step of the “reverse” trans-sulfuration pathway, the biosynthetic route that converts L-methionine to L-cysteine.83 The physiological reaction of OASS is the final step in the L-cysteine biosynthesis pathway: the reaction of O-acetyl-L-serine (OAS) and H2S to I225 S224 S70 T69 18 form L-cysteine and acetate. The reaction preceding this step is catalyzed by serine acetyltransferase, which produces OAS through the transfer of an acetyl group from acetyl-coenzyme A (CoA) to the b-hydroxyl group of L-serine.84 Figure 1.14 CBS and OASS canonical reactions. CBS and OASS also exhibit a degree of substrate promiscuity. Alternative reactions catalyzed CBS lead to the production of H2S: the reaction of L-cysteine and L-homocysteine to form L-cystathionine and H2S, the a,b-elimination of L-cysteine with H2O to produce L-serine and H2S, and the condensation of two L-cysteine molecules to produce L,L-lanthionine and H2S.85 The latter reaction can also be catalyzed by OASS in the absence of OAS.86 1.3.5 Production of lanthionine analogs Like O-acetylserine sulfhydrylase, LS can catalyze the b-replacement of O-acetyl-L-serine (OAS) and sulfide to form L-cysteine and acetate, but only when sulfide is present (refer to Figure 1.14).44 In the absence of a nucleophile, the reaction leads to the accumulation of the a-aminoacrylate intermediate. This reaction is not physiologically relevant because F. nucleatum lacks the enzyme responsible for converting L-serine to OAS (serine acetyltransferase). However, the reaction with OAS could be utilized to react different cysteine analogs with the a-aminoacrylate intermediate to generate lanthionine products for use in the synthesis of peptidoglycan fragments. As demonstrated with selenolanthionine, meso-lanthionine could be used as a meso-2,6-diaminopimelic acid bioisostere for studying peptidoglycan remodeling, biosynthesis, and sensing.87 Chemical COO-ONH2H2S+COO-HSNH2+O-acetyl-L-serine L-cysteineOO-OacetateCOO-HONH2+COO-SNH2+L-serine L-cystathionineHS COO-NH2L-homocysteineH2O-OOCNH2OASSCBS 19 synthesis of lanthionine or its analogs is reported in the literature, however, side reactions such as racemization, elimination, and Michael addition lead to low yields or tedious isolation steps.88–92 Therefore, enzymatic synthesis may be a more convenient route for the production and stereoselective synthesis of lanthionine and its derivatives. Given that lanthionine has two amino groups and two carboxylate groups, the synthesis of a lanthionine-containing peptide requires the stereocenters to be protected. However, the internal plane of symmetry within meso-lanthionine does not allow for discrimination between the amino and carboxylate groups. An alternative strategy could involve the utilization of lanthionine analogs produced by LS that mimic its structure and functionality. Therefore, using a methyl ester derivative in its place could allow us to discern between functional groups. The lanthionine synthase reactions to be investigated are with OAS and D-cysteine, D-cysteine methyl ester, or L-cysteine methyl ester to form meso-lanthionine and lanthionine methyl ester derivatives (Figure 1.15). Figure 1.15 Lanthionine analog reactions. 1.3.6 Fn1732 as a potential lanthionine epimerase F. nucleatum is unique compared to other gram-negative bacteria due to the incorporation of meso-lanthionine into the peptide crosslink of its peptidoglycan layer instead of meso-2,6-diaminopimelic acid (meso-DAP). The production of meso-DAP in bacteria that utilize this OCOO-NH2HSCOO-NH2+ SCOO-NH2-OOCNH2O-acetyl-L-serine D-cysteine meso-lanthionineO+ O-OacetateOCOO-NH2HS OONH2+ SCOO-NH2OONH2O-acetyl-L-serine D-cysteine methyl ester D,L-lanthionine methyl esterO+ O-OacetateOCOO-NH2HS OONH2+ SCOO-NH2OONH2O-acetyl-L-serine L-cysteine methyl ester L,L-lanthionine methyl esterO+ O-OacetateLSLSLS 20 diamino acid occurs in the penultimate step of the L-lysine biosynthesis pathway and involves the interconversion of L,L-DAP and meso-DAP.93 This reaction is catalyzed by DAP epimerase (encoded by dapF) and is reliant on a pair of cysteine residues that act as a thiol/thiolate pair in the active site. The reaction mechanism involves one thiolate cysteine removing a proton from L,L-DAP, and the second thiol cysteine re-protonating the Ca to generate meso-DAP (Figure 1.16).94 Given that meso-lanthionine is a thioether analog of meso-DAP, it is plausible that F. nucleatum produces meso-lanthionine in the same manner through an epimerase. An analysis of the F. nucleatum genome showed that an epimerase for the conversion of L,L-lanthionine to meso-lanthionine has not yet been identified.55 In F. nucleatum, Fn1732 is the closest homolog to dapF and may function as a lanthionine epimerase to produce meso-lanthionine for incorporation into the cell wall. Figure 1.16 DAP epimerase reaction mechanism. 1.4 Lysine 5,6-aminomutase and KamBC 1.4.1 Lysine fermentation pathway In the oral cavity, F. nucleatum serves as a bridging organism and co-aggregates with various types of bacteria through its adhesion proteins. One of these adhesion proteins, RadD, is responsible for the interactions between F. nucleatum and early colonizers such as Streptococcus and Actinomyces.95 The adhesion function of RadD is inhibited by the binding of lysine and, to a lesser extent, arginine.96 Given that lysine is one of the small subsets of amino acids utilized by F. nucleatum, its metabolic degradation may support the interspecies interactions that occur between this bacterium and early colonizers.97 Indeed, the disruption of genes within the lysine fermentation operon of F. nucleatum results in the accumulation of lysine, leading to the inhibition of RadD-mediated coaggregation with other bacteria.96 COO-NH2-OOCCysS-S CysNH2HL,L-DAPHCOO-NH2-OOCCysSH-S CysHH2Nmeso-DAPDAPepimerase 21 The lysine fermentation operon within F. nucleatum consists of several proteins that contribute to the anaerobic fermentation of L-lysine to crotonyl-CoA, a pathway first identified in Clostridium spp. (Figure 1.17).98 The first step of the lysine fermentation pathway is the interconversion of L-lysine to b-L-lysine, which is catalyzed by the PLP- and S-adenosylmethionine-dependent enzyme, lysine 2,3-aminomutase (2,3-LAM, encoded by kamA).99 This reaction involves the reductive cleavage of the C-S bond in S-adenosylmethionine to generate a 5′-deoxyadenosyl radical (Ado-CH2·) that takes part in mediating the transfer of a hydrogen from C3 of L-lysine to C2 of b-L-lysine.100 The second step of the lysine fermentation pathway involves another aminomutase to facilitate a 1,2-amino shift reaction. This enzyme, lysine 5,6-aminomutase (5,6-LAM), is the gene product of kamD and kamE and is also a PLP-dependent enzyme. Unlike 2,3-LAM, 5,6-LAM instead uses adenosylcobalamin (AdoCbl) to generate Ado-CH2· in the isomerization reaction of b-L-lysine to erythro-3,5-diaminohexanoate.101–103 The next enzyme involved in the pathway is erythro-3,5-diaminohexanoate dehydrogenase (DAHDH), the gene product of kdd, which catalyzes the NADP-dependent oxidative deamination reaction of erythro-3,5-diaminohexanoate to 3-keto-5-aminohexanoate.98 The kce gene encodes for a 3-keto-5-aminohexanoate cleavage enzyme that catalyzes the conversion of 3-keto-5-aminohexanoate and acetyl-CoA to form 3-aminobutyryl-CoA and acetoacetate.104 The production of crotonyl-CoA is carried out through the oxidative deamination reaction of 3-aminobutyryl-CoA by the gene product of kal, 3-aminobutyryl-CoA ammonia lyase.105 The lysine fermentation operon also includes a lysine permease (lysE) and a lysine riboswitch at the 5′-untranslated region that are important for controlling the catabolic flux of lysine inside the cell.55,106 The full characterization of the enzymes involved in the lysine fermentation pathway is incomplete, as the gene products for the two genes upstream from those that encode 5,6-LAM, annotated as kamB and kamC, have yet to be elucidated. 22 Figure 1.17 Lysine fermentation operon and metabolic pathway of L-lysine degradation. The genes in the lysine fermentation operon correspond to the gene numbers Fn1869-Fn1861, respectively, in F. nucleatum subsp. nucleatum (ATCC 25586). 1.4.2 Adenosylcobalamin-dependent isomerases As shown with the lysine fermentation pathway, adenosylcobalamin-dependent isomerases play an important role in catabolic pathways for amino acid degradation. Adenosylcobalamin (AdoCbl) is the biologically active form of vitamin B12. Its structure is composed of a central cobalt atom that is coordinated by a tetrapyrrole-derived macrocycle (Figure 1.18). A 5′-deoxyadenosyl group coordinates to the cobalt in the upper axial position, forming a weak organometallic bond that undergoes homolytic cleavage during a chemical reaction. The lower axial ligand that extends from the corrin macrocycle is 5,6-dimethylbenzimidazole (DMB).107,108 This covalent linkage with DMB is referred to as the “base-on” conformation. Some AdoCbl-dependent enzymes can instead form the “base-off, His-on” conformation, in which this linkage is replaced with the imidazole group of an active site histidine residue.108 Additional side chain groups including methyl, acetamide, and propionamide extend from the four pyrrole rings that form hydrogen bonding interactions with active site amino acid residues that contribute to the tight binding of the cofactor to the protein.108,109 kal kamCkce kdd kamA kamB kamD kamE lysEH2N COO-NH2H2N COO-NH2L-lysine β-L-lysineCOO-NH22,3-LAMkamANH2NADP+H2ONADPHNH3erythro-3,5-diaminohexanoateCOO-NH23-keto-5-aminohexanoateOSNH2 OCoADAHDHkddkcekaIacetyl-CoAacetoacetateSOCoA3-aminobutyryl-CoAcrotonyl-CoA5,6-LAMkamDE 23 Figure 1.18 Structure of the adenosylcobalamin cofactor. The tetrapyrrole macrocycle is shown in black, the 5′-deoxyadenosyl moiety is shown in red, and the 5,6-dimethylbenzimidazole base is shown in blue. AdoCbl-dependent isomerases catalyze 1,2-shift reactions that involve the interchange of a hydrogen atom and a substituent group, X, between adjacent carbon atoms (Figure 1.19). Depending on the enzyme, this substituent group can be a hydroxyl group, amino group, or carbon-containing fragment. The role of AdoCbl in these isomerization reactions is to serve as a radical source that allows these enzymes to catalyze reactions on otherwise unreactive molecules.110 The three subgroups of AdoCbl-dependent isomerases are the mutases, eliminases, and aminomutases. The mutases catalyze 1,2-carbon skeleton rearrangement reactions and include enzymes such as glutamate mutase, isobutyryl-CoA mutase, and methylmalonyl-CoA mutase, the only AdoCbl-dependent enzyme found in animals.111 Examples of eliminases are diol dehydratase, glycerol dehydratase, and ethanolamine ammonia lyase, which catalyze the rearrangement of 1,2-diols or N NN NCoH2NOCH2NOCH2NOCOOHOHNNNNNH2NNOOHHOOPO-OONHOCONH2CONH2CONH2 24 1,2-amino alcohols.112 The only known members of the aminomutase subgroup are ornithine 4,5-aminomutase (4,5-OAM) and lysine 5,6-aminomutase (5,6-LAM). These enzymes catalyze 1,2-amino shift reactions and require an additional PLP cofactor for catalysis.113,114 Figure 1.19 Characteristic isomerization reaction catalyzed by AdoCbl-dependent isomerases. 1.4.3 5,6-LAM reaction mechanism The reaction mechanism of AdoCbl-dependent isomerases follows the same reaction scheme. For 5,6-LAM, the internal aldimine is formed through a Schiff base linkage of the PLP cofactor and an active site lysine residue, K144 (Figure 1.20). The binding of b-L-lysine results in the displacement of the active site lysine residue and the formation of the external aldimine. Homolytic cleavage of the Co-C bond of the AdoCbl cofactor is triggered by the formation of the external aldimine, generating cob(II)alamin and a highly reactive 5′-deoxyadenosyl radical (Ado-CH2·). This radical is converted to 5′-deoxyadenosine (Ado-CH3) through proton abstraction from the C5 of b-L-lysine. Intramolecular isomerization results in the rearrangement of the substrate-derived radical to the product-like radical, which is facilitated through an intermediate radical at the C4′ position of the PLP cofactor. Abstraction of a hydrogen atom from Ado-CH3 by the C6 of the product radical results in the formation of the external aldimine with the product, erythro-3,5-diaminohexanoate. This process also regenerates Ado-CH2·, which may recombine with cob(II)alamin for another round of catalysis. The transimination reaction with K144 causes the re-formation of the internal aldimine and the release of erythro-3,5-diaminohexanoate. R2XHR1 R2HXR1X = OH, NH2, R3 25 Figure 1.20 5,6-LAM reaction mechanism. 1.4.4 The “edge-on” conformation in the structure of 5,6-LAM Lysine 5,6-aminomutase (5,6-LAM) forms an a2b2 heterodimer.114 The large a-subunit contains a triosephosphate isomerase (TIM) barrel domain, and the small b-subunit contains a Rossmann domain. Several additional a-helices and b-sheets form an accessory clamp structure at the edge of the a-subunit. Both the a- and b-subunits play a role in binding the AdoCbl and PLP cofactors; the AdoCbl cofactor is bound at the C-terminus of the Rossmann domain in the b-subunit and by the accessory clamp in the a-subunit. The PLP cofactor is bound at the C-terminus of the TIM barrel domain in the a-subunit and forms an imine linkage with a lysine residue (K144) in the Rossmann domain at the edge of the b-subunit. While other AdoCbl-dependent isomerases also contain TIM barrel and Rossman domains, the orientation of these domains within 5,6-LAM is markedly different and contributes to the mechanism of the enzyme. In the structures of glutamate mutase, methylmalonyl-CoA mutase, and diol dehydratase, the Rossmann domain is positioned NO-2-O3PONH+Lys144internal aldimineCOO-NO-2-O3PONH+external aldimineNO-2-O3PONH+NH2Lys144external aldimineβ-L-lysineH2NH2CCHHCOO-NH2NH2H2CHC COO-NH2NO-2-O3PONH2CHC COO-NH2NO-2-O3PONH+H2CHC COO-NH2NO-2-O3PONH+H3CHC COO-NH2COO-NH2NH2H2Cerythro-3,5-diaminohexanoateCo(III)H2C AdoCo(II)AdoCo(II)HAdoH2CH2CCo(III)H2C AdoCo(II)AdoCo(II)HAdoH2Csubstrate radicalproduct radical intermediate radical 26 directly above the TIM barrel domain.115–117 This forms a “top-on” conformation that positions AdoCbl close to the substrate binding site. Like the other member of the aminomutase family, 4,5-OAM, the structure of 5,6-LAM has an “edge-on” conformation where the AdoCbl cofactor is not properly positioned for catalysis (Figure 1.21 (A)).114,118 In this conformation, the Rossmann domain is tilted towards the edge of the TIM barrel domain as a result of the imine linkage formed between K144 and the PLP cofactor. This anchoring role of PLP expels AdoCbl from the active site to protect the enzyme from radical generation in the absence of substrate. Substrate binding results in the release of K144 through a transimination reaction, and thus effectively breaks the PLP-mediated anchoring interactions between the Rossmann and TIM barrel domains. This causes a change to the “top-on” conformation where AdoCbl is properly positioned in the active site, allowing for productive hydrogen transfer and substrate radical generation between the PLP-substrate complex and the AdoCbl cofactor (Figure 1.21 (B)). Figure 1.21 The “edge-on” and “top-on” conformations of 5,6-LAM. (A) Structure of 5,6-LAM in the open “edge-on” conformation (PDB entry 1XRS at 2.80 Å resolution).114 (B) Model of the closed “top-on” conformation of 5,6-LAM. The model was constructed in PYMOL (version 2.5.4) by aligning the TIM barrel and Rossmann domains with those of glutamate mutase (PDB entry 1I9C at 1.90 Å resolution).119 The TIM barrel domain is in pink, the Rossmann domain is in orange, the accessory clamp is in purple, and the PLP and AdoCbl cofactors are shown as black and red sticks, respectively. A B 27 1.4.5 Suicide inactivation in AdoCbl-dependent enzymes The mechanism of 5,6-LAM involves the 5′-deoxyadenosyl radical (Ado-CH2·) abstracting a hydrogen from an unactivated carbon-hydrogen bond in the substrate, forming 5′-deoxyadenosine (Ado-CH3). This Ado-CH3 species is not tethered to the enzyme, and therefore it is free to leave the active site. At the end of the catalytic cycle, the AdoCbl cofactor is normally regenerated through the re-formation of the Co-C bond between cob(II)alamin and Ado-CH2·. However, loss of the Ado-CH3 moiety results in the cob(II)alamin radical species being quenched through a reaction with dioxygen or the substrate/product radical intermediate, resulting in an inactive form of the cobalamin cofactor (hydroxocobalamin).120 Irreversible inactivation of the enzyme occurs because the modified cofactor remains tightly bound to the enzyme and it is unable to be exchanged with free AdoCbl.121 This substrate-induced suicide inactivation process is a shared mechanistic feature of AdoCbl-dependent isomerases.122–124 However, the AdoCbl-dependent eliminases and mutases have metallochaperone systems in place that use ATP or GTP to restore the activity of the inactivated enzyme.125,126 These accessory proteins usually form a strong operonic association and are contiguous with the genes encoding for their respective AdoCbl-dependent enzyme partner.125,127–129 As for the AdoCbl-dependent aminomutases, an analogous reactivation system has not yet been characterized. 1.4.6 KamBC as a putative metallochaperone system for 5,6-LAM In early work with 5,6-LAM in Clostridium sticklandii, it was reported that the AdoCbl-dependent enzyme system consisted of two proteins: a tightly bound cobamide protein (E1) and a sulfhydryl protein (E2).130 Further studies into the properties of 5,6-LAM (E1) denoted that the enzyme undergoes rapid inactivation that can be prevented by the addition of E2 and ATP.101 However, the gene(s) for this E2 protein as well as the mechanism by which it sustains 5,6-LAM activity have never been identified. As mentioned in Section 1.4.1, the genes kamB and kamC within the lysine fermentation operon of F. nucleatum have not been characterized. Along with the other gene products in the operon, KamB and KamC were shown to be involved in the lysine fermentation pathway as DkamB and DkamC strains of F. nucleatum led to an accumulation of lysine in the cell culture media, effects also observed with DkamA and DkamD.96 In the UniProt database, KamB is annotated as a hypothetical protein and does not share sequence homology with other proteins. 28 KamC is annotated as MutS, a DNA repair protein that contains an ATPase domain that belongs to the ABC-type of phosphate (P)-loop NTPases.131 Given these observations, it is plausible that kamB and kamC are the genes that encode for E2 and use ATP to restore the function and activity of 5,6-LAM. 1.5 Research Objectives The main goal of this thesis was to study and characterize distinct enzymes involved in the amino acid metabolism pathways within F. nucleatum. Chapter 2 focused on serine synthase, the enzyme involved in L-serine and H2S production from the reaction of L-cysteine with H2O. An aspartic acid residue (D232) acts as a general base during this forward reaction, so we wanted to investigate the possibility of D232 acting as a general acid in the reverse reaction. Chapter 3 focused on the catalytic roles of the hydrophobic residues within the serine synthase active site with respect to the general acid/base activity of D232. We also wanted to explore if mutation of these residues could expand the substrate scope of serine synthase and allow the enzyme to act as a biocatalytic tool for noncanonical amino acid synthesis. In Chapter 4, we wanted to determine if we could exploit the relaxed substrate scope of lanthionine synthase to form lanthionine analogs in reactions with cysteine derivatives. Given that lanthionine is an essential component of the cell wall within F. nucleatum, potential inhibitors were also tested with this enzyme as it would be considered a viable drug target. We also sought to discover possible routes of meso-lanthionine production in F. nucleatum through lanthionine synthase or a putative lanthionine epimerase (Fn1732). Chapter 5 investigated the role of KamB and KamC in the lysine fermentation pathway of F. nucleatum and if they function as a metallochaperone system to reactivate 5,6-LAM following substrate-induced inactivation. Given that F. nucleatum relies on these amino acid metabolism pathways for growth and survival, these objectives may provide insight into this bacterium’s metabolic adaptability in different areas of the human body. Furthermore, future research could lead to the development of drug targets to control the growth of F. nucleatum and improve the health outcomes of those with F. nucleatum-associated diseases. 29 Chapter 2: Serine Synthase 2.1 Chapter Summary Serine plays a central role in metabolism serving as a precursor for the production of amino acids, proteins, and phospholipids. Serine synthase (SS) from F. nucleatum is a fold type II PLP-dependent enzyme that generates L-serine and H2S through the b-replacement reaction of L-cysteine and H2O. Given that F. nucleatum lacks the complete pathway for the biosynthesis of L-serine, SS may play a pivotal role in maintaining L-serine homeostasis in the cell. Serine synthase may also be physiologically significant in ensuring adequate cellular L-cysteine levels, as we determined that this enzyme can also catalyze the reverse reaction. The reaction of SS with L-cysteine resulted in the transient formation of a quinonoid species, suggesting that deprotonation of the Ca and b-elimination of the thiol group from the external aldimine occurs via a stepwise mechanism. When SS was reacted with L-serine, the quinonoid species was not detected, therefore the reverse reaction likely occurs via a concerted mechanism. In the active site of SS, an aspartic acid residue (D232) was shown to play an important role in catalysis by stabilizing the more chemically reactive ketoenamine PLP tautomer and functioning as a general acid/base catalyst in the forward and reverse reactions. The substitution of D232 to an asparagine resulted in a stabilized enolimine tautomer and an inability to form neither L-serine nor L-cysteine in the forward and reverse reactions, respectively. Fluorescence experiments with SS revealed that W99, the only tryptophan residue in SS, is more solvent exposed upon substrate binding. However, the PLP cofactor is in a less polar environment, suggesting that substrate-induced closure of the active site results in W99 shielding PLP from the solvent, increasing the basicity of D232. These results highlight the importance of D232 in the catalytic mechanism of serine synthase, as well as afford more insight into the amino acid metabolism pathways within F. nucleatum. 2.2 Experimental Procedures 2.2.1 Materials F. nucleatum subsp. nucleatum (ATCC 25586) genomic DNA was from the American Tissue Culture Collection (Manassas, VA, USA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). Restriction enzymes were from New England Biolabs (Ipswich, MA, USA). PfuTurbo DNA polymerase and XL-1 Blue competent cells were from 30 Agilent Technologies (Santa Clara, CA, USA). The E.Z.N.A Plasmid DNA Mini Kit was from VWR (Radnor, PA, USA). BL21(DE3)pLysS competent cells were from EMD Biosciences (Burlington, MA, USA). The HisTrap-FF column and VivaspinÒ 20, 30 MWCO centricons were from Cytiva (Marlborough, MA, USA). Q-Sepharose High Performance resin was from GE Healthcare Technologies (Chicago, IL, USA). The PageRuler pre-stained protein ladder and the 12-14 kDa MWCO dialysis membrane tubing were from Thermo Fisher Scientific (Waltham, MA, USA). The DCTM Protein Assay was from Bio-Rad (Hercules, CA, USA). L-cysteine, L-serine, lead acetate, Na2S, NADH, and lactate dehydrogenase were from Sigma (St. Louis, MO, USA). Ninhydrin was from Alfa Aesar (Haverhill, MA, USA). All other chemicals were purchased from Thermo Fisher Scientific and were of the highest grade available. 2.2.2 Cloning and expression of serine synthase The gene encoding for serine synthase (SS), Fn1055, was amplified from the genomic DNA of F. nucleatum (ATCC 25586) with PfuTurbo DNA polymerase using the primers 5′-CA TGC CAT GGC ATG GAG CAA AAA AAA ATG AAA TAT TTA GAA AAT TTA GTT GG-3′ (forward primer) and 5′- CG CGG ATC CCG AAT TAC TCT AAG AAC ATT CTT TAT TTC-3′ (reverse primer), which contain NcoI and BamHI restriction sites (shown in bold). The PCR mixture contained PfuTurbo DNA polymerase (0.05 U/µL), PfuTurbo reaction buffer, dNTPs (200 µM), F. nucleatum genomic DNA (1.0 ng/mL), forward primer (0.25 µM), and reverse primer (0.25 µM) for a total reaction volume of 50 µL. The PCR cycle parameters were as follows: 95 °C for 2 min followed by 30 cycles of 95 °C for 30 s, 51 °C for 30 s, and 72 °C for 90 s, followed by a final 10 min extension at 72 °C. The PCR product was cleaved with NcoI and BamHI and ligated into the pET-23d vector, which was also digested with the same restriction enzymes. The ligation mixture was used to transform Escherichia coli strain XL-1 Blue competent cells, and the cell mixture was plated on Luria-Bertani (LB)-agar plates supplemented with ampicillin (100 µg/mL). The cloning strategy inserted a His6-tag onto the C-terminus of SS, and the resulting plasmid was designated pET-SS. The plasmid from the transformed cell was isolated using an E.Z.N.A Plasmid DNA Mini Kit. DNA sequencing by the Sequencing and Bioinformatics Consortium at UBC Vancouver confirmed the presence of the correct SS coding sequence in the pET-23d plasmid. pET-SS was used to transform E. coli strain BL21(DE3)pLysS for protein expression. A starter 31 culture was prepared by inoculating a single transformed colony into 100 mL of LB media supplemented with ampicillin (100 µg/mL) and chloramphenicol (34 µg/mL). The starter culture was incubated for approximately 16 h at 37 °C. Aliquots (10 mL) of the starter culture were added to 1 L of Terrific Broth (TB) supplemented with ampicillin (100 µg/mL) and chloramphenicol (34 µg/mL). The culture was incubated at 37 °C at 200 rpm until the optical density (OD) at 600 nm was ~0.6. Isopropyl b-D-1-thiogalactopyranoside (IPTG) was added to cell cultures to a final concentration of 0.2 mM. The cultures were incubated for approximately 16 h at 25 °C at 200 rpm. Cell cultures were harvested by centrifugation at 4355 g for 15 min at 4 °C, and cell pellets were pooled together and stored at -80 °C until purification. 2.2.3 Enzyme purification of serine synthase Serine synthase (SS) was purified by the following protocol. All purification steps were carried out at 4 °C. Cells overexpressing SS (15-30 g wet weight) were resuspended in 150 mL of 50 mM Tris-HCl pH 7.5 containing 5 mM imidazole and protease inhibitors benzamidine (0.5 mM) and phenylmethylsulfonyl fluoride (10 µM). The cell suspension was sonicated on ice at 30% power for 8 s at 1 min intervals for 60 min, followed by centrifugation at 39 191 g for 60 min at 4 °C. The pooled supernatant was applied to a 5 mL HisTrap-FF column equilibrated with 50 mM Tris-HCl pH 7.5 and 5 mM imidazole. Protein was eluted from the column using a step gradient: 5 column volumes (CV) of 96% Buffer A (50 mM Tris-HCl pH 7.5) and 4% Buffer B (50 mM Tris-HCl pH 7.5 and 0.5 M imidazole), 5 CV of 92% Buffer A and 8% Buffer B, 5 CV of 40% Buffer A and 60% Buffer B, and 5 CV of 100% Buffer B. Eluted protein fractions were monitored at 280 nm. Protein fractions were pooled and dialyzed against 50 mM Tris-HCl pH 7.5, 0.1 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM b-mercaptoethanol, and 10 µM PLP for 16 h at 4 °C using 12-14 kDa MWCO dialysis tubing. The protein was further applied to a Q-Sepharose High Performance column (26 x 140 mm) equilibrated with 50 mM Tris-HCl pH 7.5. The protein was eluted using a linear gradient from 100% Buffer C (50 mM Tris-HCl pH 7.5) to 100% Buffer D (50 mM Tris-HCl pH 7.5 and 0.5 M NaCl) over 8 CV. Eluted fractions of the protein were monitored at 280 nm. Fractions containing protein were pooled and concentrated to ~10 mL using a centricon with a 30 kDa MWCO filter. SDS-PAGE was performed to assess the purity of the protein. Stocks of the protein were prepared in 20% glycerol and stored at -80 °C. The 32 concentration of the apo-enzyme was determined through a Lowry assay by measuring the absorbance at 280 nm using an extinction coefficient of 6.67 mM-1 cm-1. The concentration of PLP-bound SS was determined by measuring the absorbance maximum at 410 nm using an extinction coefficient of 6.6 mM-1 cm-1. 2.2.4 Construction of serine synthase variant D232N The D232N variant of SS was created using the QuikChange approach. The forward and reverse primers for D232N were 5′-CT ACT GGA TAT AAG GTT GCT AAA CAT AGA ATA GAA GGA ATT TCT AAT GAA TTT ATA CCT GAT TTA GTA AAA TTA GAT AAA CTT GAT AAT G-3′ and 5′-C ATT ATC AAG TTT ATC TAA TTT TAC TAA ATC AGG TAT AAA TTC ATT AGA AAT TCC TTC TAT TCT ATG TTT AGC AAC CTT ATA TCC AGT AG-3′, respectively, with the mutated codon in bold. The PCR reaction mixture contained PfuTurbo DNA polymerase (0.05 U/µL), PfuTurbo reaction buffer, dNTPs (200 µM), pET-SS (10 ng), forward primer (0.25 µM), and reverse primer (0.25 µM) in a total reaction volume of 50 µL. The PCR cycling parameters were as follows: 94 °C for 2 min followed by 18 cycles at 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 5 min, followed by a final extension at 72 °C for 12 min. The PCR mixture was incubated at 37 °C for 1 h after the addition of 1 µL DpnI, followed by transformation of E. coli strain XL-1 Blue competent cells. The transformation mixture was plated on LB-agar plates supplemented with ampicillin (100 µg/mL). The plasmid from a transformed cell was isolated using an E.Z.N.A Plasmid DNA Mini Kit. The presence of the mutation and the absence of any PCR-induced errors were confirmed by DNA sequencing. The D232N variant was expressed and purified following the same protocol for wild-type SS as outlined in Section 2.2.2 and Section 2.2.3. 2.2.5 UV-visible spectroscopy Absorbance spectra of SS and D232N with L-cysteine or L-serine were measured on a PerkinElmer Lambda 25 ultraviolet (UV)-visible spectrophotometer. Spectra were recorded from 300-600 nm before and after the addition of 10 mM L-cysteine or L-serine to 50 μM of enzyme. The reactions were performed at 25 °C in a total volume of 1 mL in 100 mM HEPES-NaOH pH 7.4. 33 2.2.6 Steady-state kinetic assays measuring H2S production The steady-state rate of H2S formation by SS and D232N in the presence of L-cysteine was monitored by a continuous spectrophotometric assay that follows the reaction of H2S with lead acetate to form lead sulfide.132 The kinetic reactions were performed in triplicate at 25 °C in a total volume of 1 mL in 100 mM HEPES-NaOH pH 7.4 with 0.4 mM lead acetate. The concentration of L-cysteine was varied from 0.2 to 50 mM, and the reactions were initiated with the addition of 0.2 μM SS or 2 μM D232N. The initial velocity of the reaction was determined by the change in absorbance at 390 nm over time using an extinction coefficient of 5500 M-1 cm-1. The Michaelis-Menten equation (Equation 2.1) was fitted to a plot of the initial rate of H2S production versus the concentration of L-cysteine using a non-linear least squares regression analysis in the computer program Origin (version 8.5). Equation 2.1 v! = V"#$[S]𝐾% + [S] where vi is the initial velocity, Vmax is the maximal velocity, [S] is the substrate concentration, and KM is the Michaelis constant. The turnover number (kcat) was determined by dividing the maximal velocity by the enzyme concentration. 2.2.7 Steady-state kinetic assays measuring L-cysteine production The steady-state kinetic parameters associated with the reverse reaction, the β-replacement of L-serine with H2S to form L-cysteine and H2O, was determined through the acid-ninhydrin method.133 The reactions (1 mL) were performed in triplicate at 25 °C in 100 mM HEPES-NaOH pH 7.4 containing 50 nM SS and varying concentrations of L-serine (0.1−5 mM) and Na2S (0.1−2.5 mM). The reactions were initiated with the addition of enzyme. The Na2S concentration was fixed at 2.5 mM when the L-serine concentration was varied, and the L-serine concentration was held at 5 mM when the Na2S concentration was varied. At 1, 2, and 4 min, 200 μL aliquots of the reaction mixture were quenched with the addition of 16% (v/v) trichloroacetic acid. Glacial acetic acid (240 μL) and the acid-ninhydrin solution (240 μL) were added to the quenched solution. The reaction mixture was then heated at 100 °C for 4 min and then cooled on ice for 5 min. Cold 100% ethanol (720 μL) was then added to the solution, and the absorbance of the solution was recorded at 560 34 nm. The L-cysteine concentration was determined from a standard curve (0−150 μM) and the initial velocity was determined based on the amount of L-cysteine produced over time. To obtain kinetic parameters associated with L-cysteine production, the Michaelis-Menten equation (Equation 2.1) was fitted to a plot of the average initial velocity for each reaction versus either L-serine or HS-/H2S concentration using a non-linear least squares regression analysis in the computer program Origin (version 8.5) 2.2.8 Steady-state kinetic assays measuring pyruvate production A coupled spectrophotometric assay monitoring the NADH-dependent reduction of pyruvate to lactate by lactate dehydrogenase was used to measure the rate of pyruvate formation generated through the a,b-elimination of L-cysteine by SS or D232N. The reactions (1 mL) including 10 mM L-cysteine, 150 μM NADH, and 2 μM lactate dehydrogenase were initiated by the addition of 0.4 μM SS or D232N. The reactions were performed in 100 mM HEPES-NaOH pH 7.4 at 25 °C and recorded over 60 s. The decrease in NADH concentration was determined by the absorbance change at 340 nm using an extinction coefficient of 6.22 mM-1 cm-1. The rate of pyruvate formation by SS or D232N was determined from the slope of a linear fit following the initial lag phase using a linear regression analysis in the computer program Origin (version 8.5). 2.2.9 Stopped-flow spectroscopy Pre-steady-state kinetic analysis of the reactions between serine synthase and L-cysteine or L-serine was performed on a model SF-61DX2 stopped-flow spectrophotometer from TgK Scientific. The stopped-flow reactions were performed in 100 mM HEPES-NaOH pH 7.4 at 10 °C. A solution of enzyme (50 µM) was mixed with varying concentrations of L-cysteine or L-serine (0.5-20 mM) under the pseudo-first-order regime. The time-dependent absorbance changes in the PLP absorbance spectra were recorded at a single wavelength (410 nm or 460 nm) using a photomultiplier and at multiple wavelengths (300−700 nm) using a photodiode array detector. The absorbance traces for the single-wavelength experiments were recorded in triplicate with the average of the traces presented. Kinetic Studio (TgK Scientific) was used to extract observed rate constants by fitting a single exponential equation to the averaged absorbance traces. If the observed rate constant exhibited a hyperbolic dependence on the concentration of the amino acid, then the 35 hyperbolic function indicated in Equation 2.2 was fitted to the data using a non-linear least squares regression analysis in the computer program Origin (version 8.5). Equation 2.2 𝑘&'( = )!![+]-".[+] where kobs is the observed rate constant, Kd is the dissociation constant, [L] is the concentration of the amino acid substrate or product, and kaa is the rate constant associated with the formation of the a-aminoacrylate intermediate. If the observed rate constant had a linear dependence on the concentration of the amino acid, then the bimolecular rate constant (kon) was determined from the slope of a linear fit to the data using a linear regression analysis in the computer program Origin (version 8.5). The dissociation rate constant (koff) was calculated by multiplying the kon and Kd values as indicated in Equation 2.3. Equation 2.3 𝐾/ = 𝑘&00𝑘&1 where Kd is the dissociation constant, koff is the dissociation rate constant, and kon is the association rate constant. 2.2.10 Fluorescence spectroscopy Fluorescence spectra of SS and D232N were recorded on a PerkinElmer LS45 fluorescence spectrometer at 25 °C using a PerkinElmer PTP-1 Peltier temperature programmer. A 1 μM solution of the enzyme was mixed with 10 mM L-cysteine or L-serine in 100 mM HEPES-NaOH pH 7.4. The excitation wavelength was set to 295 nm or 320 nm, and the excitation and emission slits were set to 10 nm. Emission was measured over the wavelength range from 300 nm to 600 nm following excitation at 295 nm and from 350 nm to 600 nm following excitation at 320 nm. The fluorescence emission spectra were recorded in triplicate with the averaged spectra presented. 36 2.3 Results 2.3.1 Properties of purified serine synthase The cDNA corresponding to serine synthase (SS) was inserted into the pET-23d vector, resulting in the fusion of a His6-tag to the C-terminus of the protein. SS and the D232N variant were expressed in E. coli and purified via Ni-NTA affinity chromatography followed by anion exchange chromatography. The homogeneity and approximate size of the proteins were confirmed by SDS-PAGE. As shown in Figure 2.1, SS appeared on the gel as one band with an apparent mass of ~37 kDa, which closely matches the expected molecular weight of 37 261 Da as determined from the primary sequence. Figure 2.1 SDS-PAGE gel of purified serine synthase. SDS 12% polyacrylamide gel showing purified SS after affinity and anion exchange chromatography. Lane 1, PageRuler pre-stained protein ladder; Lane 2, purified SS after elution from the Q-Sepharose High Performance column. Purified SS appeared yellow, indicating the presence of the PLP cofactor. The PLP cofactor can exist in two tautomeric forms depending on the protonation state of the imine nitrogen; the internal proton transfer that occurs with the phenolic oxygen of the PLP cofactor results in the formation of the enolimine tautomer (deprotonated imine nitrogen) or the ketoenamine tautomer (protonated imine nitrogen) that exhibit absorbance maxima at 320 nm or 410-460 nm, respectively (Figure 2.2). 180 kDa130 kDa100 kDa70 kDa55 kDa40 kDa35 kDa25 kDaLane 1 Lane 2 37 Figure 2.2 Representation of the enolimine and ketoenamine tautomers of the PLP cofactor. The different tautomers can occur in the internal aldimine, external aldimine, and a-aminoacrylate intermediate. Experimentally determined lmax values are provided. The UV-visible absorbance spectrum of the internal aldimine of serine synthase shows a prominent lmax at 410 nm, signifying that the majority of the PLP cofactor is in the ketoenamine tautomeric form with the imine nitrogen protonated (Figure 2.3). The spectral change observed following the addition of L-cysteine was the disappearance of the peak at 410 nm and the appearance of new lmax at 320 nm and 440 nm. The peak at 320 nm likely corresponds to the formation of the enolimine tautomer of the a-aminoacrylate intermediate with the PLP imine nitrogen deprotonated. The peak at 440 nm likely represents the ketoenamine tautomer of the a-aminoacrylate intermediate, except the broadness of this peak suggests a mixture of PLP intermediates. Similarly, the addition of L-serine to the internal aldimine of SS resulted in the formation of the a-aminoacrylate species in a mixture of the enolimine and ketoenamine tautomers, as evidenced by an absorbance increase at 320 nm and a shift in lmax from 410 nm to 450 nm, respectively. NOH2-O3PONRNO-2-O3PONH+RNO2-O3PONHRenolimine (320 nm) ketoenamine (410-460 nm) 38 Figure 2.3 Absorbance spectra of serine synthase with L-cysteine and L-serine. UV-visible spectra of the PLP cofactor of 50 μM SS in its internal aldimine form (black line) and following a 2 min incubation with 10 mM L-cysteine (grey line) or L-serine (black dashed line). All 1 mL reactions were recorded in 100 mM HEPES-NaOH pH 7.5 at 25 °C. The broadness and asymmetry of the absorbance peaks formed after the addition of L-cysteine or L-serine to SS suggested a mixture of PLP intermediates, so a peak deconvolution analysis was performed using the computer program Origin (version 8.5). In the reaction of SS with L-cysteine, an analysis of the peak at 440 nm revealed an overlap of three absorbance bands with lmax values of 416, 456, and 494 nm (Figure 2.4 (A)). The peaks at 416 nm and 456 nm likely correspond to the formation of the external aldimine with L-serine and the a-aminoacrylate intermediate, respectively. The smaller peak at 494 nm represents the quinonoid species, which shows a characteristic absorbance band at 460-550 nm.134 The absorbance peak formed from the reaction of SS with L-serine at 450 nm was shown to have an overlap of two peaks at 419 nm and 460 nm, representing the external aldimine and the a-aminoacrylate intermediate, respectively (Figure 2.4 (B)). The peak at 419 nm likely corresponds to the external aldimine with L-serine and not L-cysteine because the formation of the external aldimine with L-cysteine would require nucleophilic attack of a thiol on the a-aminoacrylate intermediate, which was not present in the reaction. An equilibrium between the external aldimine with L-serine and a-aminoacrylate intermediate was formed, likely due to H2O reacting with the a-aminoacrylate intermediate. 39 Figure 2.4 Peak deconvolution analysis of serine synthase with L-cysteine and L-serine. (A) The peak at 440 nm in Figure 2.3 resulting from the reaction of 50 μM SS and 10 mM L-cysteine was deconvoluted to reveal the overlap of three peaks at 416, 456, and 494 nm. (B) The peak at 446 nm in Figure 2.3 resulting from the reaction of 50 μM SS with 10 mM L-serine was deconvoluted to reveal the overlap of two peaks at 419 nm and 460 nm. In the wild-type enzyme, the active site residue D232 participates in general acid/base chemistry to activate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. The catalytic role of D232 in the reactions with L-cysteine and L-serine was studied through the generation of a D232N variant. The substitution of this residue caused a dramatic shift in the absorbance spectrum of the internal aldimine with a major peak at 320 nm and a minor peak at 410 nm (Figure 2.5). These absorbance shifts reflect the preference for the enolimine tautomeric form of the enzyme. The addition of L-cysteine to D232N resulted in the formation of the enolimine tautomer of the a-aminoacrylate intermediate, as evidenced by a further increase at 320 nm and a shift in lmax from 410 nm to 460 nm. The formation of the a-aminoacrylate species indicates that D232 is not required for b-elimination of the thiol group from the external aldimine with L-cysteine. The reaction of L-serine with D232N led to a minor increase in absorbance at 320 nm and a significant decrease at 410 nm in comparison to the internal aldimine spectrum. This spectral change was concomitant with an absorbance shift to 422 nm, which likely indicates the formation of the enolimine tautomer of the external aldimine with L-serine. The absence of the a-aminoacrylate species suggests that the D232 acts as a general acid catalyst for the b-elimination of the hydroxyl group of L-serine. A B 40 Figure 2.5 Absorbance spectra of D232N with L-cysteine and L-serine. UV-visible spectra of 50 μM D232N (black line) and following a 2 min incubation with 10 mM L-cysteine (grey line) or L-serine (black dashed line). All 1 mL reactions were recorded in 100 mM HEPES-NaOH pH 7.5 at 25 °C. The spectra from 345-600 nm were enlarged and presented as an inset to show the formation of each intermediate more clearly. 2.3.2 Steady-state kinetic analysis of H2S production The steady-state kinetic parameters associated with H2S production by wild-type SS and D232N in the presence of L-cysteine were determined through a lead acetate assay. The initial velocity exhibited a hyperbolic dependence on L-cysteine concentration and the Michaelis-Menten equation (Equation 2.1) was fitted to the data using a non-linear least squares regression analysis (Figure 2.6). The turnover rate (kcat) of the reaction with L-cysteine for wild-type SS was determined to be 13.6 ± 0.5 s-1 with a KM(L-cys) of 1.1 ± 0.1 mM and a kcat/KM(L-cys) of 12000 ± 1800 s-1 M-1 (Table 2.1). While D232N exhibited a comparable KM(L-cys) value of 1.2 ± 0.1 mM, its kcat value (0.032 ± 0.001 s-1) was 425-fold lower than wild-type SS, resulting in a catalytic efficiency of 26 ± 4 s-1 M-1. 41 Table 2.1 Steady-state kinetic parameters associated with the production of H2S by wild-type serine synthase or D232N with increasing amounts of L-cysteine (serine synthase reaction). Enzyme kcat (s-1) KM(L-cys) (mM) kcat/KM(L-cys) (s-1 M-1) wild-type 13.6 ± 0.5 1.1 ± 0.1 12000 ± 1800 D232N 0.032 ± 0.001 1.2 ± 0.1 26 ± 4 Figure 2.6 Determination of steady-state kinetic parameters associated with H2S production for wild-type serine synthase and D232N. A plot of the initial velocity corresponding to the rate of H2S formation versus the concentration of L-cysteine for (A) wild-type SS and (B) D232N. 2.3.3 Steady-state kinetic analysis of L-cysteine production The ability of SS to catalyze the reverse reaction with L-serine and H2S to produce L-cysteine was tested using an acid-ninhydrin assay. With Na2S held at a fixed concentration, the initial rate of L-cysteine formation showed a hyperbolic dependence on L-serine concentration (Figure 2.7 (A)). A fit of the Michaelis-Menten equation (Equation 2.1) to the data using a non-linear least squares regression analysis yielded a kcat of 10.2 ± 0.3 s-1, a KM(L-ser) of 0.48 ± 0.06 mM, and a kcat/KM(L-ser) of 21000 ± 3300 s-1 M-1 (Table 2.2). Likewise, the rate of product turnover had a hyperbolic dependence on HS-/H2S concentration when L-serine concentration was fixed (Figure 2.7 (B)). This result gave kcat, KM(H2S), and kcat/KM(H2S) values of 10.3 ± 0.7 s-1, 0.27 ± 0.07 mM, and 39000 ± 13000 s-1 M-1, respectively. L-cysteine was not detected when the assay was performed under the same conditions with D232N, confirming the importance of D232 as a general acid catalyst in the cysteine synthase reaction. A B 42 Table 2.2 Steady-state kinetic parameters associated with the production of L-cysteine by serine synthase in the presence of L-serine and Na2S (cysteine synthase reaction).kcat (s-1) KM(L-ser) (mM) kcat/KM(L-ser) (s-1 M-1) KM(H2S) (mM) kcat/KM(H2S) (s-1 M-1) 10.2 ± 0.3 0.48 ± 0.06 21000 ± 3300 0.27 ± 0.07 39000 ± 13000 Figure 2.7 Determination of steady-state kinetic parameters associated with L-cysteine production for serine synthase. A plot of the initial velocity versus the concentration of (A) L-serine and (B) HS-/H2S for the reactions with SS. 2.3.4 Steady-state kinetic analysis of pyruvate production Upon formation of the a-aminoacrylate intermediate, the next catalytic step can proceed via two pathways; the first is the nucleophilic attack on the a-aminoacrylate intermediate to form the external aldimine. The alternative route is the transimination pathway, where the terminal amino group of K46 reacts with the PLP C4′ of the a-aminoacrylate intermediate to form the internal aldimine. This side reaction results in the release of 2-aminoacrylate which then undergoes non-enzymatic hydrolysis to form pyruvate and ammonia (Figure 2.8). A B 43 Figure 2.8 Transimination reaction of K46 with the a-aminoacrylate intermediate. The reaction of the side chain of K46 with the C4′ of the a-aminoacrylate intermediate leads to the formation of the internal aldimine and the release of 2-aminoacrylate. Non-enzymatic hydrolysis of 2-aminoacrylate forms pyruvate and ammonia. The rate of pyruvate formation from the transimination pathway following a,b-elimination of L-cysteine by wild-type SS or D232N was determined using a coupled spectrophotometric assay with lactate dehydrogenase. Given that lactate dehydrogenase catalyzes the NADH-dependent reduction of pyruvate to lactate, the conversion of NADH to NAD+ was monitored by following the change in absorbance at 340 nm (Figure 2.9). The concentration of NADH over the time course of the assay was determined using an extinction coefficient of 6.22 mM-1 cm-1. The first 30 s were omitted from the data analysis due to a lag phase that is characteristic of coupled enzyme assays. The rate of pyruvate formation, determined based on the initial velocity and enzyme concentration using a linear regression analysis, was 0.082 ± 0.004 s-1 for wild-type SS and 0.064 ± 0.001 s-1 for D232N (Table 2.3). Table 2.3 The rate of pyruvate production for wild-type serine synthase or D232N in the presence of L-cysteine, NADH, and lactate dehydrogenase.Enzyme Rate of pyruvate production (s-1) wild-type 0.082 ± 0.004 D232N 0.064 ± 0.001 PLPNH+COO-PLPNH+Lys46internal aldimineCOO-NH22-aminoacrylateCOO-O+ NH3pyruvateH2ONH2Lys46α-aminoacrylate 44 Figure 2.9 Rate of pyruvate production for wild-type serine synthase and D232N. A plot of NADH concentration versus time for the reaction of (A) wild-type SS or (B) D232N with L-cysteine, NADH, and lactate dehydrogenase (grey line). The initial velocity of the reaction was determined from the slope of a linear fit to the data (black line). 2.3.5 Pre-steady-state kinetic analysis with L-cysteine To obtain additional kinetic and mechanistic insight into the reactions of SS with L-cysteine or L-serine, stopped-flow experiments were performed. Rapid mixing of SS with L-cysteine resulted in multiphasic absorbance changes in the PLP cofactor. The first spectrum acquired at 2 ms showed a large increase in absorbance at 320 nm, followed by a decrease at 414 nm, indicating a decay of the internal aldimine (Figure 2.10 (A)). There was also a prominent shoulder at 490 nm, suggesting the formation of the quinonoid species. From 0.002-0.016 s, there was a continued decrease at 414 nm and an increase at 460 nm. This kinetic event likely reflects the formation of the a-aminoacrylate intermediate. The latter kinetic phases presented in Figure 2.10 (B) showed a decrease in lmax at 460 nm with a concomitant shift in lmax to 438 nm. These absorbance changes correspond to the nucleophilic attack on the a-aminoacrylate intermediate by an activated water molecule to form the external aldimine with L-serine. The final spectrum acquired at 148 s was similar to the UV-visible absorbance spectrum obtained from the reaction of SS with L-cysteine due to the reappearance of a shoulder at 490 nm (refer to Figure 2.3). A B 45 Figure 2.10 Stopped-flow spectra of serine synthase with L-cysteine. Time-resolved spectra obtained after rapid mixing of 50 μM SS with 10 mM L-cysteine (grey lines). The spectra were acquired on a log time base over 148 s and are split into two time domains: (A) 0.002-0.016 s and (B) 0.037-148 s. The black line represents the internal aldimine obtained by mixing SS with an equal volume of buffer. The arrows indicate the direction of absorbance changes over time. As shown in Figure 2.11, the reaction between D232N and L-cysteine showed a rapid decrease in lmax at 410 nm, concomitant with a small increase at 320 nm. At 0.001 s, the appearance of two small peaks at 420 nm and 490 nm indicate the formation of the external aldimine in the enolimine form, with a small fraction of the enzyme population in the quinonoid state. The presence of these intermediates was short-lived because the a-aminoacrylate intermediate was formed in less than 1 s, as denoted by an increase in absorbance at 460 nm. Unlike the wild-type enzyme, there were no further spectral changes indicating decay of the a-aminoacrylate species into another PLP intermediate. These results further support that D232 is required for the activation of a water molecule for nucleophilic attack to generate the external aldimine with L-serine. A B 46 Figure 2.11 Stopped-flow spectra of D232N with L-cysteine. Time-resolved spectra of 50 μM D232N in its internal aldimine form (black line) and following rapid mixing with 10 mM L-cysteine (grey lines). Spectra were collected between 0.001 s and 0.75 s on a log time base. The arrow represents the direction of absorbance change over time. To determine the rate constants associated with these catalytic events, single-wavelength stopped-flow experiments were performed for the reactions of wild-type SS or D232N with varying concentrations of L-cysteine (Figure 2.12). The pre-steady-state kinetic parameters associated with these events are summarized in Table 2.4. Figure 2.12 Rates associated with the formation of the external aldimine or a-aminoacrylate intermediate in the reaction of serine synthase with L-cysteine. PLPNH+COO-konkoffkaaPLPNH+COO-HSα-aminoacrylateexternal aldiminePLPNH+Lys46internal aldimineL-cys H2S 47 Table 2.4 Pre-steady-state kinetic parameters associated with the reaction between wild-type serine synthase or D232N and L-cysteine.Enzyme kon (mM-1 s-1) koff (s-1) Kd (mM) kaa (s-1) wild-type 14.8 ± 0.5 21 ± 3 1.4 ± 0.2 130 ± 6 D232N 66 ± 4 55 ± 8 0.83 ± 0.08 16.4 ± 0.4 After rapid mixing of SS with varying concentrations of L-cysteine, the absorbance traces at 410 nm were monophasic (Figure 2.13 (A)). The decrease in absorbance over time reflects the decay of the internal aldimine following substrate binding. A single exponential equation was fitted to the absorbance traces, resulting in the observed rate constant (kobs) at 410 nm displaying a linear dependence on L-cysteine concentration (Figure 2.13 (B)). The bimolecular rate constant, determined from the slope of a linear fit to the data, was 14.8 ± 0.5 mM-1 s-1. At 460 nm, the absorbance traces correspond to the formation of the a-aminoacrylate intermediate following b-elimination of the thiol group from L-cysteine (Figure 2.13 (C)). These traces were also monophasic and a single exponential equation was fitted to the data. This resulted in a hyperbolic dependence of the observed rate constant on L-cysteine concentration (Figure 2.13 (D)). A fit of Equation 2.2 to the data yielded kaa and Kd values of 130 ± 6 s-1 and 1.4 ± 0.2 mM, respectively. 48 Figure 2.13 Reaction of serine synthase with varying concentrations of L-cysteine. (A) Single-wavelength absorbance traces at 410 nm of 50 μM SS with varying amounts of L-cysteine (0.5-10 mM). (B) Linear dependence of kobs on L-cysteine measured at 410 nm. (C) Absorbance traces at 460 nm recorded after mixing 50 μM SS with varying amounts of L-cysteine (0.5-20 mM). (D) Hyperbolic dependence of kobs on L-cysteine concentration measured at 460 nm. The arrows indicate the direction of absorbance changes with increasing amounts of L-cysteine. Following the rapid mixing of D232N with L-cysteine, absorbance traces at 410 nm were monophasic and a single exponential equation was fitted to the data (Figure 2.14 (A)). The observed rate constant (kobs) displayed a linear dependence on L-cysteine concentration, producing a kon value of 66 ± 4 mM-1 s-1 (Figure 2.14 (B)). A single exponential equation was fitted to the monophasic absorbance traces obtained at 460 nm to extract values for the observed rate constant (Figure 2.14 (C)). From the plot of kobs versus L-cysteine concentration, a fit of a hyperbolic equation (Equation 2.2) to the data gave a Kd of 0.83 ± 0.08 mM and a kaa 16.4 ± 0.4 s-1 (Figure A B C D 49 2.14 (D)). These data indicate that D232 does not affect the binding of L-cysteine because the Kd value of D232N was comparable to that of the wild-type enzyme. However, in agreement with the multiwavelength absorbance traces, the variant led to an 8-fold decrease in the rate of formation of the a-aminoacrylate intermediate. Figure 2.14 Reaction of D232N with varying concentrations of L-cysteine. (A) Single-wavelength absorbance traces recorded at 410 nm following rapid mixing of 50 μM D232N and increasing amounts of L-cysteine (0.5-10 mM). (B) Linear dependence of kobs on L-cysteine concentration measured at 410 nm. (C) Absorbance traces measured at 460 nm following the reaction of D232N and varying L-cysteine concentrations (0.5-20 mM). (D) Hyperbolic dependence of kobs on L-cysteine concentration measured at 460 nm. The arrows indicate the direction of absorbance changes with increasing L-cysteine concentrations. A B C D 50 2.3.6 Pre-steady-state kinetic analysis with L-serine The changes in the PLP absorbance spectra following the reaction of SS with L-serine are shown in Figure 2.15 (A). In less than 1 ms, a rapid decrease in absorbance at 410 nm was accompanied by the appearance of a peak at 325 nm. This kinetic event likely represents the conversion of the ketoenamine tautomer of the internal aldimine to the enolimine tautomer of the external aldimine. The second kinetic phase occurred from 0.004-0.201 s where the a-aminoacrylate was formed, as evidenced by an increase in lmax at 460 nm. In comparison, rapid mixing of D232N with L-serine led to a decrease in absorbance at 410 nm and an increase at 325 nm in under 1 s (Figure 2.15 (B)). This change in absorbance likely corresponds to the conversion of the internal aldimine to the external aldimine with L-serine. However, the a-aminoacrylate species was not formed due to the absence of a peak at 460 nm. Figure 2.15 Stopped-flow spectra of wild-type serine synthase and D232N with L-serine. Time-resolved absorbance spectra of 50 μM (A) wild-type SS and (B) D232N following rapid mixing with 10 mM L-serine (grey lines). Spectra were recorded on a log time base between (A) 0.75 ms-0.201 s and (B) 0.001-0.075 s. The black line represents the internal aldimine of the enzyme when mixed with an equal volume of buffer. The arrows indicate the direction of absorbance changes over time. Single-wavelength stopped-flow experiments were repeated with wild-type SS and D232N with L-serine to determine the rates associated with the reaction (Figure 2.16). The pre-steady-state kinetic parameters are summarized in Table 2.5. Due to D232N not being able to undergo b-A B 51 elimination of the hydroxyl group on L-serine, kinetic parameters associated with the formation of the a-aminoacrylate intermediate could not be obtained for this variant. Figure 2.16 Rates associated with the formation of the external aldimine or a-aminoacrylate intermediate in the reaction of serine synthase with L-serine. Table 2.5 Pre-steady-state kinetic parameters associated with the reaction between wild-type serine synthase or D232N and L-serine.Enzyme kon (mM-1 s-1) koff (s-1) Kd (mM) kaa (s-1) wild-type 93 ± 2 67 ± 9 0.73 ± 0.08 66 ± 2 D232N 19.4 ± 0.4 ND ND ND ND – not detected As shown in Figure 2.17 (A) and (C), the absorbance traces acquired at 410 nm and 460 nm following the reaction of SS with L-serine were monophasic. Accordingly, a single exponential equation was fitted to the data. At 410 nm, the observed rate constant displayed a linear dependence on L-serine concentration, yielding a bimolecular rate constant of 93 ± 2 mM-1 s-1 (Figure 2.17 (B)). A hyperbolic fit to the observed rate constant versus L-serine concentration at 460 nm using Equation 2.2 gave a Kd of 0.73 ± 0.08 mM and a kaa of 66 ± 2 s-1 (Figure 2.17 (D)). PLPNH+COO-konkoffkaaPLPNH+COO-HOα-aminoacrylateexternal aldiminePLPNH+Lys46internal aldimineL-ser H2O 52 Figure 2.17 Reaction of serine synthase with increasing L-serine concentrations. (A) Single-wavelength absorbance traces at 410 nm after mixing 50 μM SS with L-serine (0.5-10 mM). (B) Linear dependence of kobs on L-serine concentration at 410 nm. (C) Absorbance traces acquired at 460 nm following mixing 50 μM SS with increasing L-serine concentrations (0.5-20 mM). (D) Hyperbolic dependence of kobs on L-serine concentration measured at 460 nm. The arrows indicate the direction of changes in absorbance with increasing amounts of L-serine. The conversion of the internal aldimine of D232N to the external aldimine with L-serine was followed at 410 nm. A single exponential equation was fitted to the monophasic absorbance traces, and the observed rate constants exhibited a linear dependence on L-serine concentration (Figure 2.18). The bimolecular rate constant determined from the slope of a linear fit to the data (19.4 ± 0.4 mM-1 s-1) was 3-fold lower than the value obtained for the wild-type enzyme. A B C D 53 Figure 2.18 Reaction of D232N with increasing L-serine concentrations. (A) Absorbance traces at 410 nm after mixing 50 μM D232N with L-serine (0.5-10 mM). The arrow indicates the direction of change in absorbance traces with increasing L-serine concentrations. (B) Linear dependence of kobs on L-serine concentration at 410 nm. 2.3.7 Fluorescence analysis with L-cysteine and L-serine In the UV-visible experiments, it was determined that the peak at 320 nm was attributed to the enolimine form of the PLP cofactor. However, it is possible that this peak could correspond to a gem-diamine intermediate since they absorb at the same wavelength.135,136 To distinguish between these two intermediates, the fluorescence emission spectrum was recorded after the excitation of SS at 320 nm (Figure 2.19 (A)). The addition of L-cysteine or L-serine to SS resulted in an increase in fluorescence intensity at 363 nm and the appearance of a peak at 537 nm. The peak at the shorter wavelength is characteristic of the enolimine tautomer, and the peak at the longer wavelength represents the ketoenamine tautomer.137 When the enolimine PLP tautomer is excited, it interconverts to the ketoenamine tautomer, which emits around 500 nm.137–139 As shown in the UV-visible experiments in Section 2.3.1, the addition of L-cysteine or L-serine to SS resulted in the formation of the enolimine tautomer of the PLP intermediates, which is reflected by an increase in fluorescence intensity. Therefore, the 320 nm peak observed in the UV-visible spectra after the addition of L-cysteine or L-serine is not attributed to a stable gem-diamine intermediate and instead reflects the enolimine tautomer of the a-aminoacrylate intermediate or another form of the PLP cofactor in which the imine nitrogen is deprotonated. Similar results were obtained in the fluorescence emission spectra of D232N (Figure 2.19 (B)). Excitation of the internal aldimine of A B 54 D232N resulted in emission peaks at 363 nm and 500 nm, with the former representing the enolimine tautomer and the latter representing the conversion to the ketoenamine tautomer. In the reactions with L-cysteine and L-serine, there was an increase in fluorescence intensity at 363 nm, similar to the wild-type enzyme. At the longer wavelength, the addition of L-cysteine or L-serine to D232N resulted in emission peaks with lmax values of 548 nm and 503 nm, respectively. The difference in emission wavelengths of the fluorescence spectra with D232N and L-cysteine or L-serine compared to wild-type SS is likely due to the different PLP intermediates formed upon each reaction; the reaction of D232N with L-cysteine results in the formation of the a-aminoacrylate intermediate, whereas the reaction of D232N with L-serine only forms the external aldimine with L-serine. Figure 2.19 Fluorescence emission spectra of wild-type serine synthase and D232N with L-cysteine and L-serine at 320 nm. Spectra were recorded of 1 μM (A) wild-type SS and (B) D232N in the internal aldimine form (black line) and after incubation with 10 mM L-cysteine (grey line) or L-serine (black dashed line) following excitation at 320 nm. The reactions were performed in 100 mM HEPES-NaOH pH 7.4 at 25 °C. W99 is the only tryptophan residue present in the amino acid sequence of serine synthase. To obtain information about the conformation of the enzyme and the microenvironment of the PLP cofactor, fluorescence emission was followed after excitation at 295 nm (Figure 2.20). The peak at the shorter wavelength is characteristic of the emission of a tryptophan residue (W99 in serine synthase); the peak at the longer wavelength represents the emission of the PLP cofactor. The A B 55 addition of L-cysteine or L-serine to SS caused the W99 fluorescence emission to increase and shift slightly from 338 nm to 348 nm. When tryptophan is in a nonpolar environment, it emits around 325 nm, but in more aqueous environments it emits maximally at 350 nm.140 Therefore, the red-shift of this peak indicates that following the addition of substrate, the tryptophan residue is more solvent exposed. The microenvironment of the PLP cofactor was determined based on the shift in the longer wavelength emission. A shift in the emission peak from 505 nm to 530 nm after reacting SS with L-cysteine is likely attributed to the conversion of the internal aldimine to an equilibrium mixture of the external aldimine, a-aminoacrylate intermediate, and quinonoid species. An emission peak with the same lmax was observed after the addition of L-serine to SS due to the formation of the equilibrium mixture of the external aldimine and a-aminoacrylate intermediate. The changes in fluorescence of the PLP cofactor reflect changes in its environment; the emission peak tends to occur at longer wavelengths when in less polar solvents and at shorter wavelengths when in aqueous solutions.141,142 The less polar environment of the PLP cofactor when bound to L-cysteine or L-serine may be a result of ligand-induced closure of the active site, which ends up shielding the PLP cofactor from the solvent. The large increase in intensity of this peak with both substrates is likely attributed to an increase in the efficiency of Forster resonance energy transfer (FRET) as a result of the change in the distance and/or orientation of PLP with respect to W99.138 The fluorescence spectrum for the internal aldimine of D232N exhibited emission peaks at 348 nm and 500 nm. The intensity of the shorter wavelength peak had a much larger intensity compared to the wild-type enzyme, which could be a result of D232N favouring the enolimine tautomer of the PLP cofactor. Like wild-type SS, the addition of L-serine to D232N caused a small shift in the shorter wavelength peak to 352 nm, indicating that W99 is more solvent exposed upon substrate binding. In contrast to wild-type SS, which resulted in a 25 nm red-shift following the addition of L-serine, the longer wavelength emission peak of D232N with L-serine only shifted slightly by 5 nm. The difference in magnitude of the shift is likely due to D232N only forming the external aldimine intermediate with L-serine, whereas wild-type SS forms a mixture of the external aldimine and the a-aminoacrylate intermediate. The reaction of D232N with L-cysteine resulted in a small decrease in fluorescence intensity at 348 nm and a shift in the longer wavelength from 500 nm to 550 nm, indicating that the a-aminoacrylate intermediate of the PLP cofactor is in a more nonpolar environment. The difference in the magnitude of fluorescence intensity between the spectra with L-cysteine or L-serine at the longer wavelength is likely due to the PLP intermediates being formed. 56 The reaction of D232N with L-serine displayed a higher intensity peak than for the reaction with L-cysteine because, for PLP-dependent enzymes, the a-aminoacrylate intermediate is characterized by weak fluorescence emission relative to the external aldimine.139 These fluorescence spectra support the conclusion that D232 is required for the b-elimination of the hydroxyl group in the cysteine synthase reaction with L-serine. Figure 2.20 Fluorescence emission spectra of wild-type serine synthase and D232N with L-cysteine and L-serine at 295 nm. Spectra were recorded after excitation at 295 nm of 1 μM (A) wild-type SS and (B) D232N in the internal aldimine form (black line) and mixed with 10 mM L-cysteine (grey line) or L-serine (black dashed line). The spectra were recorded in 100 mM HEPES-NaOH pH 7.4 at 25 °C. 2.4 Discussion 2.4.1 Serine synthase also functions as a cysteine synthase Serine synthase (SS) constitutes a significant enzymatic pathway for hydrogen sulfide (H2S) production in F. nucleatum. This PLP-dependent enzyme catalyzes the reaction of L-cysteine and H2O to produce L-serine and H2S. During the reaction, the formation of the a-aminoacrylate intermediate occurs following b-elimination of the thiol group of L-cysteine. The rate constant associated with this process at 10 °C (130 ± 6 s-1) was determined to be 10-fold higher than the kcat determined at 25 °C (13.6 ± 0.5 s-1), indicating that the b-elimination of the thiol group is not the rate-determining step. In comparison, Kezuka et al. estimated a similar rate of formation of the a-aminoacrylate intermediate (> 100 s-1 at 25 °C), but determined a 4-fold lower kcat value of 3.88 ± A B 57 0.04 s-1.56 The discrepancy in activity may be attributed to different buffering conditions (100 mM HEPES-NaOH pH 7.4 versus 40 mM potassium phosphate pH 7.6). Serine synthase is not unique in its ability to catalyze the reaction of L-cysteine and H2O to form L-serine and H2S. Cystathionine-b-synthase (CBS), another fold type II PLP-dependent enzyme, can also perform this b-replacement reaction. In comparison to SS, yeast CBS displays slower serine synthase activity, with a turnover number of 3.4 ± 1.0 s-1 and a KM(L-cys) of 3.6 ± 1.7 mM at 37 °C.85 Additionally, we determined that SS could also act as a cysteine synthase in the reverse reaction of L-serine and H2S to form L-cysteine and H2O. In comparison to the serine synthase reaction, the turnover number for L-cysteine production was similar at 10.2 ± 0.3 s-1, but the catalytic efficiency of the reverse reaction was 2-fold higher because of a 2-fold lower KM value. The rate of the formation of the a-aminoacrylate intermediate in the reaction with L-serine was 66 ± 2 s-1, which was 2-fold lower than with L-cysteine. This result is likely due to the hydroxyl being a poorer leaving group than the thiolate. Like the forward reaction, the formation of the a-aminoacrylate intermediate in the reverse reaction is not the rate-determining step; the slow step of the reaction might be the nucleophilic attack on the a-aminoacrylate intermediate or product release. Cysteine synthase activity is also observed in O-acetylserine sulfhydrylase (OASS), another member of the fold type II PLP-dependent enzyme family. A study by Tai et al. determined that OASS-A from Salmonella typhimurium exhibited cysteine synthase activity at 130 ± 17 s-1.143 Notably, OASS-A utilizes O-acetyl-L-serine as a substrate instead of L-serine. In addition to a 10-fold lower turnover number, SS also exhibits KM(H2S) and kcat/KM(H2S) values that are 45-fold and 560-fold lower, respectively, than OASS-A. The KM for H2S in OASS-A is 6 µM, resulting in a catalytic efficiency that approaches the limiting rate of diffusion (kcat/KM = 2.2 x 107 s-1 M-1).143 Despite the a-aminoacrylate intermediate of SS demonstrating less proficiency in reacting with H2S, cysteine synthase activity in F. nucleatum may still be physiologically relevant because H2S levels can reach up to 1.9 mM in the periodontal pocket, exceeding the apparent KM.144 58 2.4.2 Active site comparison to CBS and OASS In the forward reaction of serine synthase, the catalytic role of the active site residue D232 is to act as a general base to strengthen the nucleophilicity of a water molecule for the addition to the a-aminoacrylate intermediate.56 Given that CBS catalyzes the same reaction, we questioned if there was an equivalent active site residue in CBS. A superposition of the D232N variant of SS complexed with the a-aminoacrylate intermediate (PDB entry 5B55) and CBS complexed with the L-serine external aldimine (PDB entry 6C2Q) shows that a tyrosine residue (Y248) in the active site of CBS occupies the equivalent position of D232 in SS (Figure 2.21).56,145 Perhaps Y248 in CBS acts analogously to D232 in SS as a general acid/base to facilitate the b-replacement reaction of L-cysteine to L-serine. Figure 2.21 Superposition of SS and CBS structures. The N232 residue of the D232N variant of SS in the a-aminoacrylate form (PDB entry 5B55 at 2.14 Å resolution) is coloured grey and the Y248 residue of CBS in the external aldimine form with L-serine (PDB entry 6C2Q at 2.17 Å resolution) is coloured magenta.56,145 The carbons of the PLP cofactor complexes are green and the hydrogen bond between Y248 and L-serine is shown as a black dashed line. As mentioned in Section 2.4.1, OASS also possesses cysteine synthase activity with OAS as a substrate in place of L-serine. In the first half-reaction, the formation of the external aldimine with OAS is followed by the b-elimination of the O-acetyl group to form the a-aminoacrylate intermediate. The second half-reaction involves a nucleophilic attack of H2S onto the double bond Y248 N232 59 of the a-aminoacrylate intermediate, resulting in the formation of the external aldimine with L-cysteine. Because HS- is a good nucleophile, the cysteine synthase reaction is limited by the enzyme’s ability to form the a-aminoacrylate intermediate. Given that the O-acetyl group is a good leaving group, OASS does not require amino acid residues that act as a general acid/base to facilitate the b-elimination reaction to form the a-aminoacrylate intermediate.56 A multiple sequence alignment of SS, CBS, and OASS shows that OASS has an alanine residue in the corresponding position of D232 in SS and Y248 in CBS (Figure 2.22). Figure 2.22 Primary sequence alignment of SS with CBS and OASS enzymes from different organisms. The amino acid residues at the corresponding position of D232 in SS are shown in red. The protein sequences are as follows: Fn1055, SS from F. nucleatum (accession number: AAL95251.1), CBS_YEAST, CBS from Saccharomyces cerevisiae (accession number: P32582.1), CBS_HUMAN, CBS from Homo sapiens (accession number: P35520.2), OASS_ECOLI, OASS-A from Escherichia coli (accession number: NP_416909.1), OASS_BACSU, OASS from Bacillus subtilis (accession number: P37887.3), OASS_ MYCTO, OASS A from Mycobacterium tuberculosis (accession number: P9WP54.1), and OASS_SALTY, OASS-A from Salmonella typhimurium (accession number: P0A1E3.2). The sequence alignment was done using Clustal MUSCLE.146 2.4.3 D232 acts as an acid/base catalyst in the forward and reverse reactions of SS In serine synthase, D232 is a key active site residue for catalysis and acts through general acid/base chemistry. In this study, we generated a D232N active site variant to study the role of this residue in the b-replacement reaction of L-cysteine with L-serine. The addition of L-cysteine to D232N resulted in the formation of the a-aminoacrylate intermediate, albeit at a rate that was 8-fold lower compared to wild-type SS. Kezuka et al. similarly observed that the substitution of Asp to Asn resulted in a 4-fold decrease in the rate of a-aminoacrylate intermediate formation.56 The KM and Fn1055 G-----YKVAKHRIEGISDEFIPDLVKLDKLDNVVSVDDGDAIVMAQKLA-KCGLGVG 270 CBS_YEAST E-NLNKTDITDYKVEGIGYDFVPQVLDRKLIDVWYKTDDKPSFKYARQLISNEGVLVG 287 CBS_HUMAN E-ELNQTEQTTYEVEGIGYDFIPTVLDRTVVDKWFKSNDEEAFTFARMLIAQEGLLCG 347 OASS_ECOLI ALAGEEIKPGPHKIQGIGAGFIPANLDLKLVDKVIGITNEEAISTARRLMEEEGILAG 271 OASS_BACSU ------GKPGPHKIQGIGAGFVPDILNTEVYDEIFPVKNEEAFEYARRAAREEGILGG 265 OASS_MYCTO ------GQKGPHPIQGIGAGFVPPVLDQDLVDEIITVGNEDALNVARRLAREEGLLVG 264 OASS_SALTY ALAGEEIKPGPHKIQGIGAGFIPGNLDLKLIDKVVGITNEEAISTARRLMEEEGILAG 271 60 Kd values associated with the reaction with L-cysteine were comparable for D232N and wild-type SS, suggesting that D232 does not affect the binding of L-cysteine in the first half-reaction. However, the rate of H2S formation following a,b-elimination was 440-fold lower in D232N than in the wild-type enzyme. Although D232 is not required for b-elimination of the thiol group, it is still necessary to activate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. Given that this second half-reaction cannot occur, an accumulation of the a-aminoacrylate intermediate leads to transimination by K46, resulting in the release of 2-aminoacrylate, which then undergoes non-enzymatic hydrolysis to produce pyruvate and ammonia. The rate of pyruvate production for D232N was determined to be 0.064 ± 0.001 s-1, which was 2-fold higher than the rate of H2S formation. We also wanted to determine whether D232 acts as a general acid catalyst in the reverse reaction with L-serine. The cysteine synthase activity of SS is reliant on the formation of the a-aminoacrylate intermediate following the reaction with L-serine. While L-serine was able to bind to the D232N variant to form the external aldimine, the formation of the a-aminoacrylate intermediate did not occur. Unlike the forward reaction, the carboxylate group of D232 is required to facilitate proton-assisted b-elimination of the hydroxyl group of L-serine because it is a poorer leaving group compared to the thiol group of L-cysteine. This result was supported in the UV-visible experiments where we observed the formation of the external aldimine with L-serine as the sole PLP intermediate. Furthermore, L-cysteine was not detected in the reaction of D232N with L-serine and Na2S, confirming that D232 is required for L-cysteine production. 2.4.4 D232 stabilizes the more chemically reactive ketoenamine PLP tautomer The slower kinetic properties of D232N may also be attributed to the variant favouring the enolimine tautomer of the PLP cofactor. A shift in tautomerization from the enolimine to ketoenamine species accelerates catalysis because protonation of the imine nitrogen causes the C4′ to be more reactive towards nucleophilic attack by an amino acid substrate.147 Similarly, a protonated imine nitrogen makes the Ca proton more susceptible to deprotonation, thus facilitating subsequent b-elimination. The tautomeric equilibrium of the PLP cofactor is also strongly 61 influenced by the polarity of the active site, with the protonated ketoenamine species being stabilized in a more aqueous environment.65 Fluorescence emission spectra following excitation at 295 nm demonstrated that substrate-induced closure of the active site results in the PLP cofactor being in a more nonpolar environment. However, in the reaction with L-cysteine where both wild-type SS and D232N form the a-aminoacrylate intermediate, the PLP emission peak of wild-type SS was observed at 530 nm versus 550 nm in D232N. The red-shifted emission with D232N suggests that the PLP cofactor is in a less polar environment with respect to the wild-type enzyme. In a study with OASS-B, Speroni et al. observed that the absorbance lmax corresponding to the ketoenamine tautomer of the internal aldimine increased as a function of pH while the enolimine lmax decreased, indicating that the deprotonation of some residues in the active site stabilizes the ketoenamine species.148 Therefore, the stabilization of the ketoenamine species in wild-type SS may be a result of the negative charge of D232 creating a more polar microenvironment around the PLP cofactor. 2.4.5 The hydrophobic active site of SS may result in the stabilization of the quinonoid species The first step in the reaction with SS involves the formation of the external aldimine with the amino acid substrate, followed by deprotonation of the Ca and b-elimination from the Cb to form the a-aminoacrylate intermediate. The hydrophobic active site of SS might disfavour the accumulation of the negative charge on the Ca after proton abstraction, resulting in the delocalization of electrons through the pyridine ring to form the quinonoid species. Pre-steady-state kinetic analysis revealed that in the reaction of SS with L-cysteine, the quinonoid species appeared as a transient intermediate, suggesting that this first step occurs via a stepwise mechanism (Figure 2.23). In contrast, the quinonoid species was not detected in the reaction with L-serine, therefore the removal of the Ca proton and b-elimination of the hydroxyl group from the external aldimine with L-serine occurs through concerted steps. This result seems counterintuitive because the hydroxyl group is a poorer leaving group compared to the thiolate; however, the b-elimination of the hydroxyl group is assisted by proton donation by D232. The stability of the quinonoid species can be influenced by various factors, including the microenvironment of the active site. Perhaps the hydrophobic and restricted active site of SS slows the b-elimination of the thiolate in the reaction with L-cysteine, resulting in the formation of the quinonoid species. 62 Figure 2.23 Predicted pathways for concerted and stepwise mechanisms of serine synthase. 2.4.6 Detection of the quinonoid species in other fold type II PLP-dependent enzymes The quinonoid species has also been observed in the a2b2 system of tryptophan synthase.149 Like other members of the fold type II PLP-dependent enzyme family, tryptophan synthase forms a hydrogen bond between the pyridine nitrogen of the PLP cofactor and the b-hydroxyl group of a serine residue (S377). The interaction of the PLP pyridine nitrogen with an active site residue influences the electronic state of the cofactor, the equilibrium distribution of intermediates, and the pathway of catalysis.150 When the pyridine nitrogen is protonated, proton abstraction from the Ca favours the formation of a stabilized quinonoid species due to the delocalization of the electrons from the Ca carbanion through the pyridine ring. This was supported in a study by Jhee et al. when S377 of tryptophan synthase was mutated to an aspartic acid residue.150 Reactions with the S377D variant resulted in the accumulation of the quinonoid species, as observed through a pronounced increase in absorbance at ~500 nm. However, the substitution caused a 100-fold decrease in enzyme activity, which was suggested to be a result of the absence of active site residues to modulate the charge density associated with the aspartic acid side chain carboxylate. NO-2-O3PONH+COO-NO-2-O3PONH+COO-Xα-aminoacrylateexternal aldimineNHO-2-O3PONH+COO-Xquinonoid X = S, O 63 Notably, the quinonoid species is not an easily observed intermediate and has not been detected in other fold type II PLP-dependent enzymes like CBS, OASS, and lanthionine synthase.44,86,151 In comparison to tryptophan synthase, the unobservable quinonoid intermediate in OASS is attributed to the difference in chemical reactivity of O-acetyl-L-serine and L-serine. The good leaving group properties of acetate allow OASS to proceed via a concerted mechanism, whereas the hydroxyl group of L-serine in the external aldimine in tryptophan synthase requires activation through an intermediary step to achieve elimination.86,152 As described above, the formation of the quinonoid species is favoured when the PLP pyridine nitrogen is protonated or becomes protonated when the intermediate forms. Another reason that the formation of the quinonoid species in OASS is disfavoured is due to a mismatch in pKa values of the PLP pyridine nitrogen and an adjacent serine residue (S272), making hydrogen bond donation by S272 unlikely to occur.153 Similarly, yeast CBS has a serine residue (S289) in the equivalent position as OASS, therefore it is unsurprising that the quinonoid species does not accumulate to detectable levels in this enzyme.151 The arrangement of active site residues in CBS may also provide a plausible rationale for why the quinonoid species is not formed. The structure of CBS complexed with L-serine reveals a potential dual function of the active site lysine residue (K88).145 In addition to acting as a general base by deprotonating the Ca, the close proximity of this residue allows for electrostatic interaction between the side chain NH3+ and the negatively charged carbanion.79 This allows for shielding and stabilization of the negative charge on the Ca, thus minimizing the delocalization of charge into the conjugated ring system of the PLP cofactor to give the quinonoid species. 2.5 Concluding Remarks Serine synthase (SS) is a PLP-dependent enzyme that catalyzes the reaction of L-cysteine with H2O to form L-serine and H2S. This reaction occurs via a stepwise mechanism due to the detection of a quinonoid species. Interestingly, this PLP intermediate has not been observed in other fold type II PLP-dependent enzymes with the exception of tryptophan synthase. SS can also catalyze the reverse reaction of L-serine with H2S to form L-cysteine and H2O through a concerted mechanism. In the active site, an aspartic acid residue (D232) was determined to be a key residue for catalysis by acting as a general acid/base in both serine synthase (forward) and cysteine synthase (reverse) reactions. Although D232 is not necessarily required for b-elimination of the thiol group in the 64 forward reaction with L-cysteine, it may help to accelerate catalysis; replacement of D232 with an asparagine resulted in a 440-fold lower turnover number in comparison to the wild-type enzyme. While the a-aminoacrylate intermediate was still formed in the D232N variant, the general base catalytic properties of D232 were still needed for the activation of a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. In contrast, the a-aminoacrylate intermediate was not formed in the reverse reaction of D232N with L-serine. Therefore, D232 is also essential for proton-assisted b-elimination of the hydroxyl group from the external aldimine with L-serine. D232 also helps to boost the catalytic properties of SS through the stabilization of the ketoenamine PLP tautomer. With the imine nitrogen protonated, the energy barrier for deprotonation of the Ca is lowered and the PLP C4′ is more susceptible to nucleophilic attack. Thus, the ketoenamine tautomer enhances PLP reactivity in the elimination and addition reactions. In conclusion, the research presented herein provides insight into the multiple catalytic roles of D232 in serine synthase, a new member of the fold type II PLP-dependent enzyme family. Given the display of substrate promiscuity of these enzymes, future work could be done to further examine and expand the substrate scope of serine synthase. 65 Chapter 3: Serine Synthase Variants 3.1 Chapter Summary Serine synthase (SS) catalyzes the reversible reaction of L-cysteine and H2O to generate L-serine and H2S. The reaction mechanism involves an aspartic acid residue (D232) that functions as a general base to activate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. In the active site, D232 is surrounded by a cluster of hydrophobic residues that are key to catalysis because they increase the basicity of the carboxylate side chain. However, the presence of these aromatic side chains results in a more restricted active site and limits the number of nucleophilic substrates that can add to the a-aminoacrylate intermediate. Herein, we performed site-directed mutagenesis on four hydrophobic active site residues (W99, F125, F148, and F234) to determine their catalytic role in the serine synthase/cysteine synthase reactions (refer to Figure 1.7). We also wanted to determine if the variants could accept a broader scope of nucleophilic substrates that could react with the a-aminoacrylate intermediate. In this study, we determined that the W99A and F234A variants affected catalysis the least, while F125A and F148A had the most deleterious effects on activity. Although they could still form the a-aminoacrylate species when reacted with L-cysteine, the substitution of F125 and F148 to an alanine abolished the general base activity of D232, thus making the enzyme unable to deprotonate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. Out of the four variants, F125 and F148 are situated closest to D232 and therefore may be involved in properly positioning the side chain carboxylate for the activation of a water molecule. Overall, we determined that the W99, F125, F148, and F234 aromatic active site residues favour the forward serine synthase reaction by increasing the binding affinity of L-cysteine, decreasing the binding affinity of L-serine, and promoting the activation of a water molecule. Given that all variants formed the a-aminoacrylate intermediate in the reaction with L-serine, we tested them with a series of nucleophiles to explore the possibility of forming new b-substituted amino acids. Notably, the a-aminoacrylate intermediate of the F125A and F148A variants alone, or in combination with F234A, reacted with 2-mercaptoethanol and thiophenol, signifying that serine synthase has biocatalytic potential for the synthesis of noncanonical amino acids. 66 3.2 Experimental Procedures 3.2.1 Materials Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). DpnI was from New England Biolabs (Ipswich, MA, USA). PfuTurbo DNA polymerase and XL-1 Blue competent cells were from Agilent Technologies (Santa Clara, CA, USA). The E.Z.N.A Plasmid DNA Mini Kit was from VWR (Radnor, PA, USA). BL21(DE3)pLysS competent cells were from EMD Biosciences (Burlington, MA, USA). The HisTrap-FF column and VivaspinÒ 20, 30 MWCO centricons were from Cytiva (Marlborough, MA, USA). The 12-14 kDa MWCO dialysis membrane tubing was from Thermo Fisher Scientific (Waltham, MA, USA). The DCTM Protein Assay was from Bio-Rad (Hercules, CA, USA). L-cysteine, L-serine, lead acetate, Na2S, 4-aminophenol, 4-methoxyphenol, aniline, formic acid, indole, phenol, pyrazole, and thiophenol were from Sigma (St. Louis, MO, USA). Ninhydrin and 2-mercaptoethanol were from Alfa Aesar (Haverhill, MA, USA). All other chemicals were purchased from Thermo Fisher Scientific and were of the highest grade available. 3.2.2 Construction of SS variants Four single-site variants (W99A, F125A, F148A, and F234A) and three double-site variants (F125A/F148A, F125A/F234A, F148A/F234A) of serine synthase were produced using the QuikChange site-directed mutagenesis protocol. The PCR cycling parameters were as follows: 95 °C for 2 min following 18 cycles at 95 °C for 30 s, 54 °C for 1 min, and 72 °C for 9 min, following a final extension at 72 °C for 15 min. The forward and reverse primers for each variant are outlined in Table 3.1. 67 Table 3.1 Forward and reverse primers used in PCR to generate different SS variants. Name Sequence W99A Forward 5′-GA CAC CCT GTT ATT ATT TAT ATG CCA GAT GCG ATG AGT GAA GAA AGA AAG TCT TTA ATT CG-3′ W99A Reverse 5′-CG AAT TAA AGA CTT TCT TTC TTC ACT CAT CGC ATC TGG CAT ATA AAT AAT AAC AGG GTG TC-3′ F125A Forward 5′-G GTA AGT AGA AAA GAG GGA GGA GCT TTA GGA AGT ATA GAA AAG ACA AAA GAG TTT GC-3′ F125A Reverse 5′-GC AAA CTC TTT TGT CTT TTC TAT ACT TCC TAA AGC TCC TCC CTC TTT TCT ACT TAC C-3′ F148A Forward 5′-CT TAT TTA CCT AGC CAA GCC TCT AAC CTT TAT AAC AG -3′ F148A Reverse 5′-CT GTT ATA AAG GTT AGA GGC TTG GCT AGG TAA ATA AG-3′ F234A Forward 5′-GAA GGA ATT TCT GAT GAA GCT ATA CCT GAT TTA GTA AAA TTA G-3′ F234A Reverse 5′-C TAA TTT TAC TAA ATC AGG TAT AGC TTC ATC AGA AAT TCC TTC-3′ The mutated codons are shown in bold. DNA sequencing at UBC Vancouver confirmed the presence of the correctly inserted mutations. Each variant of SS was isolated, expressed, and purified following the same protocol for wild-type SS as outlined in Section 2.2.2 and Section 2.2.3. 3.2.3 UV-visible spectroscopy Absorbance spectra were measured on a PerkinElmer Lambda 25 UV-visible spectrophotometer. Spectra were recorded of a 50 μM solution of each enzyme variant from 300-600 nm before and after the addition of 10 mM L-cysteine or L-serine. Each 1 mL reaction was performed in 100 mM HEPES-NaOH pH 7.4 at 25 °C. 68 3.2.4 Steady-state kinetic assays measuring H2S production The production of H2S by each SS variant was monitored in a continuous spectrophotometric assay that follows the formation of lead sulfide.132 Each reaction contained 0.4 mM lead acetate and varying amounts of L-cysteine (0.5-200 mM). The kinetic reactions were initiated by the addition of enzyme (0.2 μM for W99A and F234A or 2 μM for F125A and F148A). The 1 mL reactions were performed in triplicate in 100 mM HEPES-NaOH pH 7.4 at 25 °C. The initial velocity was determined by the change in absorbance at 390 nm over 60 s using an extinction coefficient of 5500 M-1 cm-1. Kinetic parameters were obtained from a plot of the initial velocity versus L-cysteine concentration. The Michaelis-Menten equation (Equation 2.1) was fitted to the data using a non-linear least squares regression analysis in the computer program Origin (version 8.5). 3.2.5 Steady-state kinetic assays measuring L-cysteine production The kinetic parameters associated with cysteine synthase activity were measured using the acid-ninhydrin method.133 Each 1 mL reaction was performed in triplicate at 25 °C and contained 2.5 mM Na2S and varying amounts of L-serine (0.1-5 mM) in 100 mM HEPES-NaOH pH 7.4. The kinetic reactions were initiated by the addition of 50 nM W99A, 1 μM F125A or F148A, or 0.2 μM F234A. The reaction mixture was quenched with the addition of 16% (v/v) trichloroacetic acid at different time points (1, 2, and 4 min for W99A, and 1, 5, 10, and 15 min for F125A, F148A, and F234A). Glacial acetic acid (240 μL) and the acid-ninhydrin solution (240 μL) were added to the quenched solution. The solution was heated at 100 °C for 4 min and then cooled on ice for 5 min. Cold 100% ethanol (720 μL) was added to each solution. The absorbance for each reaction mixture was recorded at 560 nm. The concentration of L-cysteine formed from each reaction was determined based on a standard curve (0-150 μM) and the average initial velocity was determined based on the amount of L-cysteine produced over time. Kinetic parameters were determined by fitting the Michaelis-Menten equation (Equation 2.1) to the initial velocity versus L-serine concentration data using a non-linear least squares regression analysis in the computer program Origin (version 8.5). 69 3.2.6 Stopped-flow spectroscopy Pre-steady-state kinetic analysis of the reactions between the SS variants and L-cysteine or L-serine was performed on a model SF-61DX2 stopped-flow spectrophotometer from TgK Scientific. The stopped-flow reactions of each SS variant (20 µM or 50 µM) with varying amounts of L-cysteine (0.3-200 mM) or L-serine (0.3-20 mM) were performed in 100 mM HEPES-NaOH pH 7.4 at 10 °C under the pseudo-first-order regime. The time-dependent changes in the PLP absorbance spectra were recorded at a single wavelength (410 nm or 460 nm) using a photomultiplier and at multiple wavelengths (300−700 nm) using a photodiode array detector. A single exponential equation was fitted to the averaged absorbance traces from the single-wavelength stopped-flow experiments using Kinetic Studio (TgK Scientific) to extract observed rate constants. If the observed rate constant (kobs) exhibited a hyperbolic dependence on the concentration of the amino acid substrate, then a hyperbolic function was fitted to the data as indicated in Equation 2.2 using a non-linear least squares regression analysis in the computer program Origin (version 8.5). If the observed rate constant showed a linear dependence on the concentration of the amino acid substrate, then the bimolecular rate constant (kon) was determined from the slope of a linear fit to the data using a linear regression analysis and the dissociation rate constant (koff) was determined as indicated in Equation 2.3 using the computer program Origin (version 8.5). 3.2.7 Fluorescence spectroscopy Fluorescence emission spectra of the SS variants were recorded on a PerkinElmer LS45 fluorescence spectrometer at 25 °C using a PerkinElmer PTP-1 Peltier temperature programmer. Each 3 mL reaction contained 1 μM of enzyme mixed with 10 mM L-cysteine or L-serine in 100 mM HEPES-NaOH pH 7.4. Each enzyme variant was excited at 295 nm and 320 nm, and the emission was recorded from 300-600 nm and 350-600 nm, respectively. Both excitation and emission slits were set to 10 nm. 3.2.8 Absorbance assays with other nucleophiles Absorbance spectra were recorded from 350-550 nm using a PerkinElmer Lambda 25 UV-visible spectrophotometer. A solution of the enzyme (20 μM wild-type SS or variants) was incubated for 5 min with 10 mM L-serine in 100 mM HEPES-NaOH pH 7.4 at 25 °C. Spectral changes were 70 observed after further addition of 10 mM 2-mercaptoethanol, 4-aminophenol, 4-methoxyphenol, aniline, formic acid, indole, phenol, pyrazole, or thiophenol. 3.3 Results 3.3.1 Properties of SS variants Each of the SS variants (W99A, F125A, F148A, and F234A) were purified by Ni-NTA affinity and anion exchange chromatography. Like the wild-type enzyme, the purified variants exhibited a major absorbance peak at 410 nm, indicating that the internal aldimine of the PLP cofactor was primarily the ketoenamine tautomer with the imine nitrogen protonated (Figure 3.1). The spectra of the internal aldimine of F148A and F234A also showed a minor peak at 320 nm that represents the enolimine tautomer of the PLP cofactor. The addition of L-cysteine to W99A resulted in the formation of peaks at 320 nm and 426 nm with a broad shoulder at 490 nm. Reactions of F148A or F234A with L-cysteine showed similar results, with an increase at 320 nm and a shift in absorbance to 418 nm with a shoulder at 475 nm for F148A or 490 nm for F234A. These shifts in absorbance indicate that L-cysteine can bind to the enzyme variants, but the asymmetric nature of the peaks in the visible range suggests a mixture of different PLP intermediates. The peaks with lmax values at 418-422 nm and 475-490 nm likely represent the external aldimine and the quinonoid species, respectively; a full analysis of the deconvoluted peaks is discussed below to confirm the identity of the intermediates formed. The reaction of L-cysteine with F125A led to the formation of the a-aminoacrylate intermediate, as evidenced by a lmax at 460 nm. Similar to the other variants, there was also an absorbance increase at 320 nm, indicating a mixture of the enolimine and ketoenamine tautomers of the a-aminoacrylate species. The absence of a peak around 420 nm corresponding to the L-serine external aldimine suggests that F125A abolished the general base activity of D232, preventing deprotonation of a water molecule for attack on the a-aminoacrylate intermediate. In the reactions of the SS variants with L-serine, there was less variability in the PLP intermediates formed. The addition of L-serine to W99A or F234A resulted in the formation of a peak at 320 nm and a second broad band centered at 450 nm. These spectra are similar to the spectrum obtained of wild-type SS with L-serine, suggesting that these variants also formed an equilibrium mixture of the external aldimine with L-serine and the a-aminoacrylate intermediate. In contrast, the reactions of L-serine with F125A or F148A resulted in a narrower 71 peak at 460 nm, indicating only formation of the a-aminoacrylate intermediate. These spectra indicate that while F125A and F148A allow for b-elimination of the hydroxyl group, they are unable to activate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate, preventing establishment of an equilibrium mixture with the L-serine external aldimine. Figure 3.1 Absorbance spectra of SS variants with L-cysteine and L-serine. UV-visible absorbance spectra of 50 μM (A) W99A, (B) F125A, (C) F148A, and (D) F234A in the internal aldimine form (black line) and following a 30 min incubation with 10 mM L-cysteine (grey line) or L-serine (black dashed line). All spectra were recorded in 100 mM HEPES-NaOH pH 7.4 at 25 °C. The broadness in some of the absorbance peaks prompted us to perform a peak deconvolution analysis using the computer program Origin (version 8.5) to determine the PLP intermediates formed in the reactions of the SS variants with L-cysteine or L-serine. The peak with a lmax of 422 A B C D 72 nm in the reaction of W99A with L-cysteine revealed the overlap of three peaks at 426, 466, and 490 nm (Figure 3.2 (A)). These peaks correspond to the formation of the external aldimine with L-serine and/or L-cysteine, the a-aminoacrylate intermediate, and the quinonoid species, respectively. This result is similar to that obtained from the peak analysis of wild-type SS with L-cysteine. Peak analysis of the absorbance spectra recorded after the reactions of F148A or F234A with L-cysteine showed absorbance peaks at 422 nm and 478 nm (F148A) or 482 nm (F234A), signifying the external aldimine with L-cysteine and the quinonoid species, respectively (Figure 3.2 (B, C)). These results suggest that these variants do not form the a-aminoacrylate intermediate due to an absence of a lmax at 460 nm. However, stopped-flow data in Section 3.3.4 revealed that the reactions of F148A or F234A with L-cysteine show transient formation of the a-aminoacrylate intermediate. Perhaps the excess bisulfide released from the b-elimination of the thiol group in the spectral assay above readily reacts with the a-aminoacrylate intermediate, resulting in a final equilibrium mixture of the quinonoid species and the external aldimine with L-cysteine. The reactions of W99A or F234A with L-serine produced a broad peak with a lmax at 450 nm. The peak deconvolution analysis revealed that the broad absorbance band was a mixture of two peaks with lmax at ~420 nm and ~460 nm, representing the external aldimine with L-serine and the a-aminoacrylate intermediate, respectively (Figure 3.2 (D, E)). An equilibrium mixture of these intermediates likely occurs because D232 is able to activate a water molecule that can add to the a-aminoacrylate intermediate. Therefore, W99A and F234A retain the activity of D232 to act as a general base to activate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. 73 Figure 3.2 Peak analysis of SS variants with L-cysteine and L-serine. Peak analysis of the UV-visible absorbance spectra in Figure 3.1 resulting from the reactions of 50 μM (A, D) W99A, (B) F148A, and (C, E) F234A with 10 mM L-cysteine (top panels) or L-serine (bottom panels). The cumulative peaks (black) were deconvoluted to reveal the overlap of spectra arising from different PLP intermediates (grey). 3.3.2 Steady-state kinetic analysis of H2S production The steady-state kinetic parameters associated with H2S production from the reactions of the SS variants with L-cysteine were determined through a continuous spectrophotometric assay (Table 3.2). As determined from the UV-visible experiments, the substitution to an alanine in the F125A and F148A variants resulted in the apparent inability of D232 to activate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. Therefore, the forward reactions of these variants do not produce L-serine. This was reflected in the H2S assay where the kcat of F125A was 0.110 ± 0.001 s-1, which was 124-fold lower than the wild-type enzyme. By comparison, the KM for L-cysteine was 2-fold lower in F125A than wild-type SS, resulting in a 60-fold lower catalytic efficiency. In contrast, the KM for F148A was much higher than wild-type SS to the point that we were unable to reach saturation at high concentrations of L-cysteine (200 mM). This resulted in a 1200-fold decrease in catalytic efficiency of F148A compared to the wild-type enzyme, with a A B C D E 74 kcat/KM of 10.0 ± 0.1 s-1 M-1. Given that the b-elimination of the thiol group still occurs to form the a-aminoacrylate intermediate due to its properties of being a good leaving group, the low rates of H2S formation associated with these variants are likely due to the enzyme proceeding through the transimination pathway, as illustrated in Figure 3.3. In this alternative pathway, the side chain of K46 reacts with the PLP C4′ of the a-aminoacrylate intermediate to form the internal aldimine and release 2-aminoacrylate as a product. In contrast, W99A and F234A were able to utilize the general base activity of D232 to activate a water molecule in the normal catalytic cycle, as evidenced by an equilibrium mixture of the L-serine external aldimine and the a-aminoacrylate intermediate in the presence of excess L-serine. Therefore, enzyme turnover determined from the H2S assay with W99A or F234A and excess L-cysteine likely indicates L-serine formation (Figure 3.4). The W99A variant exhibited a 2-fold decrease in both the kcat and KM values, resulting in a comparable kcat/KM to wild-type SS. The catalytic efficiency of the F234A variant was 6-fold lower than wild-type SS due to a 9-fold and 1.5-fold decrease in the kcat and KM values, respectively. Figure 3.3 Catalytic cycle of the serine synthase reaction with L-cysteine. The reaction of the SS variants and L-cysteine results in the b-elimination of H2S and the formation of the a-aminoacrylate intermediate. The addition of H2O to the a-aminoacrylate intermediate leads to L-serine formation in W99A and F234A. The reaction pathway indicated by a dashed arrow shows the transimination reaction observed for the F125A and F148A variants. This reaction results in the release of 2-aminoacrylate and the re-formation of the internal aldimine. PLPNH+COO-PLPNH+COO-HSα-aminoacrylateexternal aldiminePLPNH+Lys46internal aldimineL-cysH2SPLPNH+COO-HOexternal aldimineL-ser2-aminoacrylateH2OF125AF148A 75 Table 3.2 Steady-state kinetic parameters associated with H2S production by SS variants in the presence of lead acetate and L-cysteine.Enzyme kcat (s-1) KM(L-cys) (mM) kcat/KM (s-1 M-1) wild-type1 13.6 ± 0.5 1.1 ± 0.1 12000 ± 1800 W99A 6.4 ± 0.2 0.46 ± 0.07 13800 ± 2600 F125A 0.110 ± 0.001 0.55 ± 0.03 200 ± 15 F148A ND ND 10.0 ± 0.1 F234A 1.45 ± 0.05 0.75 ± 0.09 1900 ± 300 1 – from Table 2.1 ND – not determined Figure 3.4 Determination of steady-state kinetic parameters associated with H2S production for SS variants. A plot of the initial velocity versus L-cysteine concentration was generated for the reactions of (A) W99A, (B) F125A, (C) F148A, and (D) F234A with lead acetate and varying amounts of L-cysteine. A B C D 76 3.3.3 Steady-state kinetic analysis of L-cysteine production The steady-state kinetic parameters associated with L-cysteine production by the SS variants were determined through a discontinuous acid-ninhydrin assay. As shown in Table 3.3 and Figure 3.5, each of the variants was able to produce L-cysteine, albeit with different levels of enzyme activity. Similar to the H2S assay, F125A and F148A exhibited the lowest levels of activity with comparable kcat and KM values that resulted in catalytic efficiencies that were 80-fold lower compared to wild-type SS. The W99A variant had a 2-fold lower kcat (4.9 ± 0.2 s-1) and a 2-fold higher KM (0.9 ± 0.1 mM) compared to the wild-type enzyme, resulting in a 4-fold lower catalytic efficiency (5650 ± 910 s-1 M-1). The kcat/KM value associated with the reaction of L-serine and Na2S with F234A was 8-fold lower than wild-type SS due to a 2.5-fold lower kcat of 3.91 ± 0.09 s-1 and 3-fold higher KM of 1.5 ± 0.1 mM. Table 3.3 Steady-state kinetic parameters associated with L-cysteine production by SS variants in the presence of L-serine and Na2S.Enzyme kcat (s-1) KM(L-ser) (mM) kcat/KM (s-1 M-1) wild-type1 10.2 ± 0.3 0.48 ± 0.06 21000 ± 3300 W99A 4.9 ± 0.2 0.9 ± 0.1 5650 ± 910 F125A 0.179 ± 0.005 0.65 ± 0.07 274 ± 37 F148A 0.167 ± 0.006 0.63 ± 0.08 265 ± 42 F234A 3.91 ± 0.09 1.5 ± 0.1 2600 ± 230 1 – from Table 2.2 77 Figure 3.5 Determination of steady-state kinetic parameters associated with L-cysteine production for SS variants. A plot of the initial velocity versus L-serine concentration was generated for the reactions of (A) W99A, (B) F125A, (C) F148A, and (D) F234A with a fixed concentration of Na2S and varying concentrations of L-serine. 3.3.4 Pre-steady-state kinetic analysis with L-cysteine Using a stopped-flow system, spectral changes of the PLP cofactor were recorded following the rapid mixing of the SS variants with L-cysteine or L-serine. The addition of L-cysteine to each variant resulted in the formation of the a-aminoacrylate intermediate as evidenced by a decrease in absorbance at 410 nm and an increase at 460 nm (Figure 3.6). The lower absorbance according to the a-aminoacrylate intermediate in the spectra of W99A and L-cysteine with respect to the other variants is likely attributed to the released bisulfide or activated water that can readily react with the a-aminoacrylate intermediate to form an equilibrium of PLP intermediates, which were observed over longer time domains. A B C D 78 Figure 3.6 Stopped-flow spectra of SS variants with L-cysteine. Time-resolved spectra of 50 µM (A) W99A, (B) F125A, (C) F148A, and (D) F234A following rapid mixing with 10 mM L-cysteine (grey lines). Spectra were acquired on a log time base from (A) 0.002-0.152 s, (B) 0.002-0.083 s, (C) 0.001-0.104 s, and (D) 0.001-0.128 s. The arrows indicate the direction of the absorbance changes over time. The black line represents the internal aldimine of each variant. To determine the rates associated with the binding and release of L-cysteine and the rate associated with the formation of the a-aminoacrylate intermediate, single-wavelength stopped-flow traces were acquired at 410 nm and 460 nm (Table 3.4). A B C D 79 Table 3.4 Pre-steady-state kinetic parameters for the reactions of SS variants with L-cysteine.Enzyme kon (mM-1 s-1) koff (s-1) Kd (mM) kaa (s-1) wild-type1 14.8 ± 0.5 21 ± 3 1.4 ± 0.2 130 ± 6 W99A 6.5 ± 0.2 138 ± 17 21 ± 2 240 ± 8 F125A 11.5 ± 0.2 429 ± 29 37 ± 2 450 ± 9 F148A 1.59 ± 0.03 82 ± 4 52 ± 1 84 ± 1 F234A 2.96 ± 0.08 312 ± 45 106 ± 12 350 ± 25 1 – from Table 2.4 For all SS variants, the absorbance traces at 410 nm were monophasic and a single exponential equation was fitted to the data. The observed rate constant (kobs) showed a linear dependence on L-cysteine concentration to give bimolecular rate constants ranging from 1.59 ± 0.03 mM-1 s-1 to 11.5 ± 0.2 mM-1 s-1, which were lower than the kon value observed for wild-type SS. A single exponential equation was fitted to the monophasic absorbance traces measured at 460 nm. The kobs showed a hyperbolic dependence on L-cysteine concentration and the corresponding Kd and kaa values were obtained from a fit of Equation 2.2 to the data. For all four variants, the Kd values increased 15- to 80-fold, with the F234A variant having the highest Kd value of 106 ± 12 mM. Compared to wild-type SS, a 2- to 3-fold increase in kaa was observed for W99A, F125A, and F234A, while the F148A variant exhibited a 1.5-fold lower kaa value. All single-wavelength absorbance traces and plots of the observed rate constant versus L-cysteine concentration for each SS variant are shown in Figure 3.7-Figure 3.10. 80 Figure 3.7 Reaction of W99A with increasing concentrations of L-cysteine. Single-wavelength absorbance traces at (A) 410 nm and (C) 460 nm following the reaction of 20 µM W99A mixed with L-cysteine (0.3-200 mM). The arrows indicate the direction of absorbance changes with increasing L-cysteine concentrations. (B) Linear dependence of kobs on L-cysteine concentration at 410 nm. (D) Hyperbolic dependence of kobs on increasing amounts of L-cysteine at 460 nm. A B C D 81 Figure 3.8 Reaction of F125A with increasing concentrations of L-cysteine. Absorbance traces at (A) 410 nm and (C) 460 nm following rapid mixing of 20 µM F125A with L-cysteine (0.3-200 mM). The arrows for the absorbance traces indicate the direction of changes in absorbance with increasing L-cysteine concentrations. (B) Linear dependence of kobs on L-cysteine concentration at 410 nm. (D) Hyperbolic dependence of kobs on L-cysteine concentration at 460 nm. A B C D 82 Figure 3.9 Reaction of F148A with increasing concentrations of L-cysteine. Single-wavelength absorbance traces at (A) 410 nm and (C) 460 nm after rapid mixing of 20 µM F148A with varying concentrations of L-cysteine (0.3-200 mM). The arrows indicate the direction of changes in absorbance with increasing L-cysteine concentrations. (B) Linear dependence of kobs on L-cysteine concentration at 410 nm. (D) Hyperbolic dependence of kobs on L-cysteine concentration at 460 nm. A B C D 83 Figure 3.10 Reaction of F234A with increasing concentrations of L-cysteine. Single-wavelength absorbance traces acquired at (A) 410 nm and (C) 460 nm following the reaction of 20 µM F234A with varying amounts of L-cysteine (0.3-200 mM). The arrows indicate the direction of absorbance changes with increasing L-cysteine concentrations. (B) Linear dependence of kobs on L-cysteine concentration at 410 nm. (D) Hyperbolic dependence of kobs on L-cysteine concentration at 460 nm. 3.3.5 Pre-steady-state kinetic analysis with L-serine Stopped-flow experiments were also performed with the reactions of the SS variants and L-serine. The resulting spectra were similar to the spectra acquired in the reactions with L-cysteine; following rapid mixing of each variant with L-serine, a decrease in absorbance at 410 nm was followed by an increase in absorbance at 460 nm, indicating a-aminoacrylate intermediate formation (Figure 3.11). Notably, the W99A and F234A variants formed the a-aminoacrylate C D B A 84 intermediate within 0.3 s, whereas the F125A and F148A variants formed the intermediate much slower, over 23 and 4.37 s, respectively. Figure 3.11 Stopped-flow spectra of SS variants and L-serine. Time-resolved absorbance spectra of 50 µM (A) W99A, (B) F125A, (C) F148A, and (D) F234A in the internal aldimine form (black lines) and following rapid mixing with 10 mM L-serine (grey lines). Spectra were acquired on a log time base from (A) 0.002-0.31 s, (B) 0.002-23 s, (C) 0.001-4.37 s, and (D) 0.001-0.29 s. The arrows indicate the direction of absorbance changes over time. Single-wavelength absorbance traces were also acquired in the reactions of the SS variants with L-serine. The kinetic parameters associated with the decay of the internal aldimine and the formation of the a-aminoacrylate intermediate at 410 nm and 460 nm, respectively, are summarized in Table 3.5. A B C D 85 Table 3.5 Pre-steady-state kinetic parameters for the reactions of SS variants with L-serine.Enzyme kon (mM-1 s-1) koff (s-1) Kd (mM) kaa (s-1) wild-type1 93 ± 2 67 ± 9 0.73 ± 0.08 66 ± 2 W99A 38 ± 1 14 ± 2 0.37 ± 0.05 13.5 ± 0.4 F125A 4.12 ± 0.07 1.4 ± 0.4 0.35 ± 0.09 0.17 ± 0.01 F148A 1.80 ± 0.05 0.76 ± 0.06 0.42 ± 0.02 0.240 ± 0.003 F234A 26.2 ± 0.5 4.4 ± 0.4 0.17 ± 0.01 2.89 ± 0.04 1 – from Table 2.5 Like the wild-type enzyme, all absorbance traces at 410 nm and 460 nm were monophasic and a single exponential equation was fitted to the data. At 410 nm, each variant showed a linear dependence of the observed rate constant (kobs) on L-serine concentration, giving kon rates that ranged from 1.80 ± 0.05 mM-1 s-1 to 38 ± 1 mM-1 s-1. Similar to the reactions with L-cysteine, all of the kon rates were lower in the variants compared to the wild-type enzyme. The observed rate constants acquired at 460 nm showed a hyperbolic dependence on L-serine concentration and Equation 2.2 was fitted to the data. In contrast to the reactions with L-cysteine, the L-serine reactions with each SS variant gave Kd values that were 2- to 4-fold lower than the wild-type enzyme, consequently giving lower koff values as well. There was also a more dramatic effect on kaa with L-serine as a substrate, with F125A and F148A exhibiting 400-fold and 300-fold lower values, respectively, compared to wild-type SS. The W99A and F234A variants exhibited a 5-fold and 25-fold reduction in a-aminoacrylate intermediate formation, respectively. The absorbance traces recorded and plots of the observed rate constant versus L-serine concentration at 410 nm and 460 nm are shown in Figure 3.12-Figure 3.15. 86 Figure 3.12 Reaction of W99A with increasing concentrations of L-serine. Absorbance traces at (A) 410 nm and (C) 460 nm were acquired following the reaction between 20 µM W99A with varying concentrations of L-serine (0.3-20 mM). The arrows indicate the direction of changes in absorbance with increasing concentrations of L-serine. (B) Linear dependence of kobs on L-serine concentration at 410 nm. (D) Hyperbolic dependence of kobs on L-serine concentration at 460 nm. A B C D 87 Figure 3.13 Reaction of F125A with increasing concentrations of L-serine. Absorbance traces recorded following rapid mixing of 20 µM F125A with L-serine (0.3-20 mM) at (A) 410 nm and (C) 460 nm. The arrows indicate the direction of absorbance changes with increasing L-serine concentrations. (B) Linear dependence of kobs on L-serine concentration at 410 nm. (D) Hyperbolic dependence of kobs on L-serine concentration at 460 nm. C D B A 88 Figure 3.14 Reaction of F148A with increasing concentrations of L-serine. Single-wavelength absorbance traces were acquired following the reaction of 20 µM F148A with L-serine (0.3-20 mM) at (A) 410 nm and (C) 460 nm. The arrows indicate the direction of absorbance changes with increasing amounts of L-serine. (B) Linear dependence of kobs on L-serine concentration at 410 nm. (D) Hyperbolic dependence of kobs on L-serine concentration at 460 nm. B A C D 89 Figure 3.15 Reaction of F234A with increasing concentrations of L-serine. Absorbance traces were acquired following the reaction of 20 µM F234A with L-serine (0.3-20 mM) at (A) 410 nm and (C) 460 nm. The arrows indicate the direction of absorbance changes with increasing L-serine concentrations. (B) Linear dependence of kobs on L-serine concentration at 410 nm. (D) Hyperbolic dependence of kobs on L-serine concentration at 460 nm. 3.3.6 Fluorescence analysis with L-cysteine and L-serine Fluorescence experiments with each SS variant were performed with an excitation wavelength at 320 nm to confirm that the corresponding peak observed in the UV-visible experiments was attributed to the enolimine tautomer of the PLP cofactor rather than a stabilized gem-diamine intermediate. As shown in Figure 3.16 (A), excitation of the internal aldimine of W99A at 320 nm resulted in emission maxima at 363 nm and 425 nm, which represent the excited states of the enolimine and ketoenamine tautomers, respectively.139 The addition of L-cysteine or L-serine to W99A resulted in the appearance of a new emission peak around 541 nm. This peak represents the C D B A 90 enolimine tautomer of the a-aminoacrylate intermediate which undergoes interconversion to the ketoenamine species when in the excited state. As shown in Figure 3.16 (B, C, and D), excitation of the internal aldimine of F125A, F148A, or F234A at 320 nm resulted in emission peaks at 363 nm and 430 nm (F125A), 424 nm (F148A), or 422 nm (F234A). The reactions of these variants with L-cysteine or L-serine showed an increase in fluorescence intensity with a lmax at 545 nm (F125A and F234A) or 548 nm (F148A). These fluorescence spectra were similar to W99A, which is likely due to the formation of the same PLP intermediates. As evidenced by the stopped-flow experiments, the addition of L-cysteine or L-serine to the variant enzymes resulted in the formation of the a-aminoacrylate species. The increased intensity of the longer wavelength peak observed in F125A and F148A is likely attributed to these variants forming primarily the a-aminoacrylate species, whereas W99A and F234A form an equilibrium mixture of PLP intermediates. These results indicate that, like wild-type SS, the peak at 320 nm in the absorbance spectra of the SS variants corresponds to the formation of the enolimine tautomer of the different PLP intermediates. 91 Figure 3.16 Fluorescence emission spectra of SS variants with L-cysteine and L-serine at 320 nm. Spectra of 1 μM (A) W99A, (B) F125A, (C) F148A, and (D) F234A following excitation at 320 nm. The black line represents the internal aldimine form of the enzyme and the grey and black dashed lines are the spectra recorded after incubation with 10 mM L-cysteine and L-serine, respectively. All reactions were performed in 100 mM HEPES-NaOH pH 7.4 at 25 °C. The F125A, F148A, and F234A variants were also excited at 295 nm to observe the influence of these aromatic residues on the PLP microenvironment. As described in Section 2.3.7, the fluorescence emission at the shorter and longer wavelengths correspond to the environments around W99 and the PLP cofactor, respectively. Excitation of W99 at 295 nm in F125A resulted in emission peaks at 335 nm and 495 nm (Figure 3.17 (A)). In comparison to wild-type SS, the shorter wavelength emission peak is 3 nm blue-shifted, suggesting that W99 is in a more nonpolar environment. The 10 nm blue-shift in the longer wavelength peak suggests that the PLP cofactor is in a more polar environment. The addition of L-cysteine or L-serine to F125A did not cause a A B C D 92 change in the emission wavelength or intensity at 335 nm. However, there was a shift from 495 nm to 545 nm, concomitant with a dramatic increase in fluorescence intensity. The 50 nm red-shifted peak represents the conversion of the PLP cofactor to the a-aminoacrylate intermediate as well as a more hydrophobic environment around PLP, likely due to W99 shielding PLP from the solvent in the closed conformation. In F148A and F234A, excitation at 295 nm led to an emission lmax at 342 nm, which is 7 nm red-shifted compared to F125A (Figure 3.17 (B, C)). This indicates that W99 is in a more polar environment in F148A and F234A in comparison to F125A. The emission spectra recorded after the addition of L-cysteine or L-serine resulted in the appearance of a peak at 542 nm in F148A and 537 nm in F234A. The decrease in intensity in the F234A spectra may be due to a decrease in the energy transfer between W99 and PLP as a result of W99 being more solvent exposed. Given that each variant forms the a-aminoacrylate intermediate after the reaction with L-cysteine or L-serine, the lmax of the longer emission wavelengths gives more insight into the degree of polarity around the PLP cofactor. A more blue-shifted peak in F234A suggests that out of the three variants, it is in the most polar environment, followed by F148A and then F125A. Figure 3.17 Fluorescence emission spectra of SS variants with L-cysteine and L-serine at 295 nm. Spectra were recorded after excitation at 295 nm of 1 μM (A) F125A, (B) F148A and (C) F234A before (black line) and after mixing with 10 mM L-cysteine (grey line) and L-serine (black dashed line). The spectra were recorded in 100 mM HEPES-NaOH pH 7.4 at 25 °C. 3.3.7 Reaction of SS variants with other nucleophiles Given that wild-type SS and each SS variant was able to form the a-aminoacrylate intermediate in the reaction with L-serine, we explored the possibility of serine synthase catalyzing b-A B C 93 replacement reactions with other nucleophiles (Figure 3.18). The nucleophiles tested were as follows: 2-mercaptoethanol, 4-aminophenol, 4-methoxyphenol, aniline, formic acid, indole, phenol, pyrazole, and thiophenol. A list of the nucleophiles is shown in Table 3.6. Figure 3.18 Reaction of a nucleophile with the a-aminoacrylate intermediate to form a new b-substituted amino acid. Table 3.6 List of tested nucleophiles and their respective structures.Name Structure Name Structure 2-mercaptoethanol indole 4-aminophenol phenol 4-methoxyphenol pyrazole aniline thiophenol formic acid PLPNH+COO-Nuinternal aldimineexternal aldiminePLPNH+α-aminoacrylateNuCOO-COO-NH2NuPLPNH+Lys46HSOHHNOHH2NOHOHH3CONHNNH2 SHH OHO 94 To test the ability of SS to catalyze these b-replacement reactions with different nucleophiles, the a-aminoacrylate intermediate was first formed by reacting the enzyme with L-serine (Figure 3.19). Following the formation of the a-aminoacrylate intermediate (lmax at 450 nm), the addition of each nucleophile to wild-type SS did not induce a shift in the PLP absorbance spectra, which is likely due to the restrictive nature of the hydrophobic active site. Due to the proximity of the phenylalanine residues to the PLP cofactor, we tested the nucleophilic addition reactions with the F125A, F148A, and F234A variants, as well as double mutants of F125A/F148A, F125A/F234A, and F148A/F234A. With each of these single-site or double-site variants, the reactions with L-serine resulted in the formation of the a-aminoacrylate intermediate, as evidenced by an absorbance band with a lmax at 444-460 nm. As shown in Figure 3.20, the F125A/F148A variant formed the external aldimine when reacted with L-serine (lmax at 423 nm). Given that the a-aminoacrylate intermediate was not formed in this reaction, the F125A/F148A variant was excluded from the reactions with other nucleophiles. The addition of 2-mercaptoethanol to the a-aminoacrylate intermediate in F125A, F148A, F125A/F234A, or F148A/F234A resulted in a shift in absorbance to 423-439 nm, indicating nucleophilic attack of the thiol group on the a-aminoacrylate species. Despite having a more solvent exposed active site, the reaction of 2-mercaptoethanol with F234A only caused a small shift in absorbance to 443 nm. This small change in absorbance may be due to this variant retaining the general base properties of D232, allowing water to react with the a-aminoacrylate intermediate to form an equilibrium mixture with the L-serine external aldimine. In all variant enzymes, the addition of thiophenol to the a-aminoacrylate intermediate resulted in a shift in absorbance to 420-423 nm, suggesting the formation of the external aldimine with the product. The other nucleophiles that were tested did not exhibit any spectral changes denoting product formation, suggesting that the a-aminoacrylate intermediate is more efficient in reacting with thiol-based nucleophiles. 95 Figure 3.19 Absorbance spectra of SS variants with different nucleophiles. UV-visible absorbance spectra of 20 µM (A) wild-type SS, (B) F125A, (C) F148A, (D) F234A, (E) F125A/F234A, and (F) F148A/F234A mixed with 10 mM L-serine (black line). Spectral changes were observed following the addition of 10 mM 2-mercaptoethanol (grey line) or thiophenol (grey dashed line). All spectra were recorded in 100 mM HEPES-NaOH pH 7.4 at 25 °C. Figure 3.20 Absorbance spectra of F125A/F148A with L-serine. UV-visible spectrum of 20 µM F125A/F148A following incubation with 10 mM L-serine. The 1 mL reaction was performed in 100 mM HEPES-NaOH pH 7.4 at 25 °C. A B C D E F 96 3.4 Discussion 3.4.1 Mutation of hydrophobic residues in SS affects the acid/base activity of D232 The catalytic mechanism of serine synthase involves the use of an aspartic acid residue, D232, to act as a general acid/base to facilitate catalysis. In the active site, D232 is surrounded by a cluster of hydrophobic residues that increase the basicity of the carboxylate side chain, thus increasing the catalytic proficiency of the enzyme. In this study, we substituted four of these residues (W99, F125, F148, and F234) with an alanine to determine their catalytic role and influence on the general acid/base activity of D232. In the reaction with L-cysteine, the formation of the a-aminoacrylate intermediate results from proton abstraction from the Ca and b-elimination of the thiol group from the L-cysteine external aldimine. The reaction proceeds to the L-serine external aldimine when D232 deprotonates a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. Given that the thiol group is a good leaving group, it is unsurprising that all the SS variants were able to form the a-aminoacrylate intermediate when reacted with L-cysteine. In the b-elimination reaction, the variant with the least affected substitution was W99A, which had a comparable catalytic efficiency to wild-type SS due to a 2-fold decrease in the kcat and KM values. The F234A variant had a 6-fold reduction in catalytic efficiency compared to wild-type SS due to a 9-fold decrease in kcat and a 1.5-fold decrease in KM. The biggest decrease in activity occurred in the F125A and F148A variants. The low turnover rates of F125A and F148A in the H2S assay suggested that the reaction proceeds through the transimination pathway with K46 displacing 2-aminoacrylate from the a-aminoacrylate intermediate to form the internal aldimine. Substitution of F125 and F148 to an alanine resulted in the inability of D232 to act as a general base in the activation of a water molecule. Consequently, these variants could no longer function as a serine synthase. In contrast, the W99A and F234A variants retained the activity of D232, which resulted in a more modest decrease in enzyme activity for the forward reaction. The carboxylate side chain of D232 is also important in the reverse reaction by facilitating proton-assisted b-elimination of the hydroxyl group from the L-serine external aldimine. Similarly to the forward reaction, each variant was able to form the a-aminoacrylate intermediate in the reaction with L-serine. However, in comparison to wild-type SS, the rate associated with the formation of the a-aminoacrylate species was 400-fold and 300-fold lower in F125A and F148A, respectively. 97 The rate of L-cysteine production by these variants was also 60-fold lower than wild-type SS, whereas the W99A and F234A variants only led to a 2- to 3-fold decrease in activity. The more pronounced effects of the b-elimination activity with L-serine in F125A and F148A compared to L-cysteine may be because of the hydroxyl group being a poorer leaving group compared to the thiolate. However, the general acid properties of D232 are required for b-elimination of the hydroxyl group side chain of L-serine. Perhaps the smaller side chains of F125A and F148A affect the orientation of D232 in the active site, and thus its ability to effectively act as a general acid in the b-elimination of the hydroxyl group. 3.4.2 The positioning of active site residues in SS may affect catalytic performance When looking at the distance between the side chains of the aromatic residues and the carboxylate side chain of D232, it is unsurprising that the residues closest to D232 had the most pronounced effect on the catalytic performance of the enzyme. For example, W99A had the least effect on enzyme activity and is consequently ~7.3 Å from the D232 carboxylate side chain. The F234A variant, located ~6 Å away from D232, had the second lowest effect on SS activity. The most deleterious substitutions were in F125A and F148A, as shown by a pronounced decrease in activity in both the forward and reverse reactions. These residues are positioned ~3.5-3.8 Å from D232. The deleterious effects of F125A and F148A may also be a result of the interactions that may occur between their aromatic side chains and the carboxylate side chain of D232 to properly position it for catalysis. In enzymes, the active site is a distinguishing feature where noncovalent interactions are used to precisely position enzymatic functional groups with respect to the substrate or other amino acid residues for efficient catalysis. One example of a traditional noncovalent interaction involved with the stabilization of protein structure and protein-ligand interactions is hydrogen bonding. However, in the active site of serine synthase, there are no hydrogen bond contacts surrounding D232. A newer type of noncovalent interaction that has been studied with other proteins is an anion-aromatic pair, where a negatively charged side chain interacts with the partially positively charged edge of an aromatic ring. The analysis of different proteins in the Protein Data Bank revealed that aspartic acid and glutamic acid residues show a preference towards an interaction with phenylalanine side chains.154–156 This type of interaction is observed in the 98 active site of bacterial ketosteroid isomerase (KSI) from Comamonas testosteroni. In the active site of KSI, an aspartic acid residue (D38) functions as a general base by deprotonating C4-H of the steroid substrate, thus generating a dienolate intermediate.154 There are no hydrogen bonding groups adjacent to D38, but there are two phenylalanine residues (F54 and F116) situated near each of the oxygen atoms of the aspartic acid carboxylate side chain.157,158 The proximity of these residues results in favourable anion-aromatic interactions between D38 and the ring edges of F54 and F116 that ensures the proper orientation of D38 for catalysis, as well as the binding of hydrophobic substrates (Figure 3.21 (A)).154,159 The positioning of F54 and F116 with respect to D38 in KSI is reminiscent of F125 and F148 with D232 in serine synthase. However, it appears that only F148 has an edgewise interaction with the carboxylate side chain of D232 (Figure 3.21 (B)).56 Based on this structural information, it is likely that the deleterious effects of F148A on enzymatic activity may be a result of the incorrect positioning of D232 based on the disruption of favourable anion-aromatic interactions. Figure 3.21 Anion-aromatic interactions in KSI and SS. Structures of (A) ketosteroid isomerase (KSI; PDB entry 1OCV at 2.00 Å resolution) and (B) serine synthase (SS; PDB entry 5Z5C at 2.07 Å resolution) showing anion-aromatic interactions between the active site aspartic acid and phenylalanine residues.56,159 The structure obtained for KSI was for a F116W variant, but the residue was changed back to a Phe in PYMOL (version 2.5.4). The carbons of the amino acid side chains are coloured grey and the PLP cofactor in SS is coloured green. The noncovalent interactions between the carboxylate of Asp and the ring edge of Phe are shown as a black dashed line. A B F116 F54 D38 F125 F148 D232 99 3.4.3 The hydrophobic active site cluster of SS favours the forward reaction with L-cysteine The hydrophobic environment of serine synthase (SS) also affects the binding affinities of L-cysteine and L-serine. In the reaction with L-cysteine, wild-type SS exhibited a Kd value of 1.4 ± 0.2 mM. Pre-steady-state kinetic analysis showed that all SS variants had a weaker binding affinity for L-cysteine with values ranging from 21 ± 2 mM to 106 ± 12 mM. In contrast, L-serine bound to the variants with slightly lower binding affinities compared to wild-type SS (0.73 ± 0.08 mM), exhibiting Kd values ranging from 0.17 ± 0.01 mM to 0.42 ± 0.02 mM. The variations in binding affinities for these amino acid substrates are likely due to their differing polarities. The substitution of the aromatic residues to an alanine causes an increased polarity of the active site, thus favouring the more polar hydroxyl side chain of L-serine. The tighter binding affinity for L-cysteine in wild-type SS is likely a result of a lower koff rate, which would be expected with a more nonpolar side chain around the hydrophobic side chains in the active site. Therefore, the cluster of hydrophobic residues in the SS active site favours the forward reaction by increasing the binding affinity of the substrate, L-cysteine, and decreasing the binding affinity of the product, L-serine. 3.4.4 SS variants have biocatalytic potential in the synthesis of noncanonical amino acids The reaction mechanism of serine synthase involves the b-elimination of an amino acid substrate to form an a-aminoacrylate intermediate. The electrophilicity of the a-aminoacrylate intermediate allows for the potential of nucleophilic substrates to be incorporated as amino acid side chains to form new amino acids. The development of these noncanonical amino acids (ncAAs) has been demonstrated in tryptophan synthase, another fold type II PLP-dependent enzyme. Like serine synthase, the b-subunit of tryptophan synthase catalyzes the b-elimination of L-serine to form the a-aminoacrylate intermediate. In the physiological reaction, indole, generated from the cleavage of indole-3-glycerol phosphate (IGP) by the a-subunit, adds to the a-aminoacrylate intermediate in the b-subunit to form L-tryptophan. In addition to C-C bond formation, tryptophan synthase can form C-S or C-N bonds by catalyzing b-substitution reactions with a variety of indole analogs or other non-indole nucleophiles.160,161 Given that serine synthase also forms a stable a-aminoacrylate intermediate when reacted with L-serine, we wanted to explore the possibility of this enzyme being able to react with different nucleophiles to form new b-substituted amino acids. 100 In order to test the biocatalytic potential of serine synthase in ncAA synthesis, the a-aminoacrylate intermediate formed from the reaction with L-serine was reacted with a variety of nucleophilic substrates. Given the small, hydrophobic nature of the active site, the nucleophiles able to react with the a-aminoacrylate intermediate in wild-type SS were limited to water and bisulfide. However, when the hydrophobic active site residues were substituted with an alanine, we were still able to form the a-aminoacrylate intermediate when reacted with L-serine. This result allowed us to test the nucleophilic addition reactions with the SS variants. The fluorescence experiments showed that out of the three phenylalanine variants, F234A displayed the highest degree of polarity around W99 and the PLP cofactor. We thought that the more solvent-exposed active site of F234A compared to F125A and F148A would allow for better access of bulky nucleophiles. Interestingly, the a-aminoacrylate intermediate generated by F125A, F148A, or F234A reacted with thiophenol, but only F125A and F148A reacted with the smaller substrate 2-mercaptoethanol. The ability of F234A to retain the general acid/base catalytic properties of D232 may have caused excess H2O to react with the a-aminoacrylate intermediate and consequently prevent nucleophilic attack of 2-mercaptoethanol. In contrast, the increased electron density around the sulfur atom in thiophenol makes it a better nucleophile than the hydroxide ion generated by D232, thus allowing for addition to the a-aminoacrylate intermediate. Overall, it was determined that F125A and F148A alone, or in combination with F234A, provided the most reactivity with the tested nucleophiles. Although the reactions with the SS variants were limited to S-nucleophiles, they provide a starting point from which to expand the substrate scope of SS and its biocatalytic potential through directed evolution. 3.5 Concluding Remarks The cluster of aromatic residues (W99, F125, F148, and F234) surrounding D232 in the active site of serine synthase are important in catalysis because they increase the basicity of the side chain of D232 and thus, catalytic efficiency towards proton abstraction. The substitution of these residues to an alanine resulted in an overall decrease in activity, with the W99A and F234A variants exhibiting the most modest difference compared to wild-type SS. The F125A and F148A variants are closest to D232 in the active site, and consequently caused the most negative effects on catalytic performance. These residues abolished the forward serine synthase reaction due to their 101 inability to activate a water molecule for nucleophilic attack on the a-aminoacrylate intermediate. Inspection of the active site showed that the carboxylate side chain of D232 forms an anion-aromatic interaction with the partially positively charged ring edge of F148, which may help position D232 in the active site for general acid/base catalysis. To test the potential functional connection between F148 and D232, a double mutant cycle similar to that generated for ketosteroid isomerase (KSI) could be implemented with a mutation of D232 to a glutamic acid residue.154 The addition of a methylene group to the side chain of D232 would result in mispositioning of the general base, which usually causes deleterious effects compared to the wild-type enzyme.162,163 If F148 was involved in positioning D232 for catalysis, then there should be similar deleterious effects when comparing the activities of the already mispositioned base in the D232E and F148A/D232E variants. The hydrophobic active site in SS also restricts the nucleophilic substrates that can enter the binding pocket to react with the a-aminoacrylate intermediate. The F125A and F148A variants, along with the double mutants F125A/F234A and F148A/F234A, were able to bind 2-mercaptoethanol and thiophenol to presumably generate the external aldimine with the noncanonical amino acid product. These products could be further isolated and characterized by nuclear magnetic resonance (NMR) spectroscopy to confirm their identity. Additional mutations could be introduced to the amino acid sequence of serine synthase through directed evolution to further investigate its potential in the synthesis of noncanonical amino acids. 102 Chapter 4: Lanthionine Synthase 4.1 Chapter Summary Most gram-negative bacteria incorporate meso-2,6-diaminopimelic acid (meso-DAP) into the peptidoglycan layer of their cell wall, but F. nucleatum substitutes this diamino acid with meso-lanthionine. Meso-lanthionine is produced from two equivalents of L-cysteine, and this reaction is catalyzed by the PLP-dependent enzyme lanthionine synthase (LS). We utilized the relaxed substrate scope of LS and established that D-cysteine, D-cysteine methyl ester, and L-cysteine methyl ester could be added to the a-aminoacrylate intermediate of wild-type LS or a S224A variant. The production of lanthionine analogs from these reactions could be used in the production of peptides for peptidoglycan biosynthesis, remodeling, and sensing. We also explored possible mechanisms for enzymatic production of meso-lanthionine. The reaction of LS with L-cysteine and D-cysteine resulted in a mixture of L,L-lanthionine and meso-lanthionine, with a preference for the latter diastereomer. Compared to L-cysteine, pre-steady-state kinetics showed that D-cysteine reacts with the a-aminoacrylate intermediate with a kon that was 2-3-fold faster and a Kd that was 2-3-fold lower than that of the L-isomer. The other possible route for meso-lanthionine production was through an epimerase. In the genome of F. nucleatum, Fn1732 is the closest homolog to a DAP epimerase, which racemizes L,L-DAP to meso-DAP. A coupled spectrophotometric assay revealed that Fn1732 can convert L,L-lanthionine to meso-lanthionine with a kcat of 0.070 ± 0.001 s-1 and a KM of 1.8 ± 0.1 mM. Due to having a low turnover rate, Fn1732 may not encode for a lanthionine epimerase and may instead act as a racemase on another amino acid. A series of DAP and cysteine analogs were tested as inhibitors of LS. The inhibition studies revealed that meso-DAP, a bioisostere of meso-lanthionine, is a more potent inhibitor than its diastereomer, L,L-DAP. Given that lanthionine synthase is an important enzyme for meso-lanthionine production and F. nucleatum viability, it could be considered a viable drug target to control the growth of F. nucleatum. 4.2 Experimental Procedures 4.2.1 Materials Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). F. nucleatum subsp. polymorphum (ATCC 10593) genomic DNA was purchased from the American 103 Tissue Culture Collection. Q5Ò High Fidelity DNA polymerase and restriction enzymes were from New England Biolabs (Ipswich, MA, USA). XL-1 Blue competent cells and the C-18 column were from Agilent Technologies (Santa Clara, CA, USA). The E.Z.N.A Plasmid DNA Mini Kit and 10 kDa MWCO microcons were from VWR (Radnor, PA, USA). BL21(DE3) competent cells were from EMD Biosciences (Burlington, MA, USA). The HisTrap-FF column and VivaspinÒ 20, 30 MWCO centricons were from Cytiva (Marlborough, MA, USA).The PageRuler pre-stained protein ladder and the 12-14 kDa MWCO dialysis membrane tubing were from Thermo Fisher Scientific (Waltham, MA, USA). L-cysteine, D-cysteine, NADP+, lead acetate, dansyl chloride, lanthionine (L,L- and meso- mixture), D,L-2-aminopimelic acid, S-(2-aminoethyl)-L-cysteine, S-(2-boronoethyl)-L-cysteine, S-allyl-L-cysteine, L,L-2,6-diaminopimelic acid, meso-2,6-diaminopimelic acid, and meso-oxa-2,6-diaminopimelic acid were from Sigma (St. Louis, MO, USA). O-acetyl-L-serine, D-cysteine methyl ester, L-cysteine methyl ester, D,L-2-aminosuberic acid, and S-(2-carboxyethyl)-L-cysteine were from TCI Chemicals (Portland, OR, USA). Recombinant meso-diaminopimelic acid dehydrogenase from Corynebacterium glutamicum was obtained from Michael Murphy’s group from UBC Vancouver.164 All other chemicals were purchased from Thermo Fisher Scientific and were of the highest grade available. 4.2.2 Cloning and expression of lanthionine synthase and Fn1732 The gene associated with lanthionine synthase (Fn1220) was previously cloned and expressed from F. nucleatum (ATCC 25586) genomic DNA and inserted into the pET-15b vector through a published protocol.44 The gene encoding for the putative lanthionine epimerase (AT688_RS01580) was PCR amplified from the genomic DNA of F. nucleatum subsp. polymorphum (ATCC 10953) using Q5â High-Fidelity DNA polymerase. The primers were as follows: 5′-G GAA TTC CAT ATG GAT AAA AAA GTG CAA GTT TTA G-3′ (forward primer) and 5′-GCG GAT CCG ATT TAA TAT ATA CTT TAC CCT CAG C-3′ (reverse primer) with the restriction sites shown in bold (NdeI and BamHI, respectively). The PCR mixture contained Q5 DNA polymerase (0.02 U/µL) Q5 reaction buffer, dNTPs (200 µM), F. nucleatum subsp. polymorphum genomic DNA (1.0 ng/mL), forward primer (0.5 µM), and reverse primer (0.5 µM) for a total reaction volume of 50 µL. The PCR cycle parameters were as follows: 98 °C for 30 s followed by 30 cycles of 98 °C for 10 s, 67 °C for 30 s, and 72 °C for 30 s. The PCR product was 104 cleaved with NdeI and BamHI for subcloning the cDNA into the same restriction sites of pET-26b. The ligation mixture was then used to transform E. coli strain XL-1 Blue competent cells, and the cell mixture was plated on LB-agar plates supplemented with kanamycin (30 µg/mL). The plasmid construct pET-LanE was isolated using an E.Z.N.A Plasmid DNA Mini Kit and the presence of the correct coding sequence was confirmed by Sanger sequencing. pET-LanE was used to transform E. coli strain BL21(DE3) for protein expression. A single colony from LB-agar plates supplemented with kanamycin (30 µg/mL) was used to inoculate a 100 mL LB starter culture that was then incubated at 37 °C for 16 h. Aliquots (10 mL) of the starter culture were added to 1 L of 2YT media supplemented with kanamycin (30 µg/mL) and the culture was incubated at 200 rpm at 37 °C. When the OD600 reached 0.6, IPTG (0.2 mM) was added to the culture, and then it was further incubated for approximately 16 h at 25 °C at 200 rpm. Cells were harvested by centrifugation at 4355 g for 15 min at 4 °C, and the cell pellets were pooled together and stored at -80 °C. 4.2.3 Enzyme purification of lanthionine synthase and Fn1732 Lanthionine synthase was purified previously following a published protocol.44 Fn1732 was purified by the following protocol with all steps carried out at 4 °C. The bacterial cell pellet (15 g) was resuspended in 35 mL lysis buffer (50 mM Tris-HCl pH 7.5, 0.15 M NaCl, 1 mM b-mercaptoethanol) with 5 mM imidazole, 0.5 mM benzamidine, and 10 µM phenylmethylsulfonyl fluoride. The cells were further lysed by sonication at 25% power for 8 s at 1 min intervals for 60 min, followed by centrifugation at 39 191 g for 1 h at 4 °C. The supernatant was applied to a 5 mL HisTrap-FF column equilibrated with lysis buffer. The column was washed with 100 mL lysis buffer with 20 mM imidazole and then eluted with 100 mL lysis buffer containing 300 mM imidazole. The eluted protein was dialyzed against 50 mM Tris-HCl pH 7.5, 0.15 M NaCl, and 5 mM b-mercaptoethanol for 16 h at 4 °C using 12-14 kDa MWCO dialysis tubing. SDS-PAGE was performed to assess protein purity. The concentration of the protein was determined by the absorbance value at 280 nm using an extinction coefficient of 26 360 M-1 cm-1 that was calculated from the amino acid sequence using the ExPASy ProtParam tool.165 Protein stocks were prepared in 20% glycerol and stored at -80 °C. 105 4.2.4 Construction of lanthionine synthase variant S224A The S224A variant of LS was cloned through site-directed mutagenesis, expressed, and purified following a previously published protocol.166 4.2.5 UV-visible spectroscopy UV-visible absorbance spectra were measured on a PerkinElmer Lambda 25 UV-visible spectrophotometer. Spectra were recorded from 300-600 nm of 50 μM LS with 1 mM O-acetyl-L-serine (OAS) before and after the addition of 1 mM D-cysteine or D-cysteine methyl ester, and 10 μM S224A with 1 mM OAS before and after the addition of 1 mM L-cysteine methyl ester. Each 1 mL reaction was performed in 100 mM HEPES-NaOH pH 7.4 at 25 °C. 4.2.6 Steady-state kinetic assays measuring lanthionine production The ability of LS to form meso-lanthionine or D,L-lanthionine methyl ester was monitored through a coupled spectrophotometric assay using diaminopimelic acid dehydrogenase (DAPDH). DAPDH catalyzes the oxidative deamination of meso-2,6-diaminopimelic acid (meso-DAP) to form L-2-amino-6-ketopimelic acid and ammonia. The reaction is dependent on the reduction of NADP+ to NADPH. The kinetic assays (1 mL) were performed in triplicate in 100 mM HEPES-NaOH pH 7.4 at 25 °C . The reaction mixture contained 0.1 mM NADP+, 2 μM DAPDH, 5 mM OAS, and varying amounts of D-cysteine or D-cysteine methyl ester (0.01-2 mM). The reactions were initiated by the addition of 0.2 μM LS. The initial velocity of the reaction was determined by the absorbance change associated with NADPH formation over 5 min at 340 nm using an extinction coefficient of 6.22 mM-1 cm-1. To measure the possible lanthionine epimerase activity of Fn1732, L,L-lanthionine was first synthesized through the reaction of 20 μM LS and 50 mM L-cysteine in 10 mL of 100 mM Tris-HCl pH 8.6 with 1 mM b-mercaptoethanol. The reaction mixture was incubated for 2 h at 37 °C in the fume hood. The enzyme was removed by centrifugation using a centricon with a 30 kDa MWCO filter. The rate of formation of meso-lanthionine by Fn1732 was monitored using the same coupled spectrophotometric kinetic assay with DAPDH. The reaction mixture contained 0.1 mM NADP+, 1 mM dithiothreitol (DTT), 1 mM EDTA, 2 μM DAPDH, and varying amounts of L,L-lanthionine (0.1-20 mM). The reactions (1 mL) were performed in triplicate in 100 mM Tris-HCl pH 8.6 at 37 °C and initiated with 1 μM Fn1732. 106 The initial velocity of the reaction was determined by the change in absorbance from 50-150 s at 340 nm following the initial lag phase as described above. Kinetic parameters were determined by fitting the Michaelis-Menten equation (Equation 2.1) to the data using a non-linear least squares regression analysis in the computer program Origin (version 8.5). 4.2.7 High-performance liquid chromatography (HPLC) The production of different lanthionine products was confirmed using reversed-phase HPLC. A reaction mixture (1 mL) containing 1 mM L-cysteine in 100 mM HEPES-NaOH pH 7.4 was initiated by the addition of 1 µM LS and the reaction mixture was incubated at 37 °C for 2 h. The enzyme was then filtered out by centrifugation using microcon filters (10 kDa cut-off). Following this step, 500 µL of a dansyl chloride solution in acetonitrile (1.5 mg/mL) was added to the reaction mixture. The mixture was then covered in foil to avoid light and was incubated for 30 min at room temperature. Following incubation, 100 µL of 2% ethylamine was added to the mixture and filtered through a 0.2 µM syringe filter. Aliquots (10 µL) of each sample were injected onto a C-18 column (Agilent Poroshell 120, 4.6 x 100 mm), and the dansylated products were separated at a flow rate of 0.5 mL/min at 40 °C with a mobile phase consisting of 33.25/66.75% (v/v) methanol/water, 0.6% (v/v) glacial acetic acid, and 0.008% (v/v) triethylamine. The appearance of the dansylated products was monitored through absorbance changes at 338 nm over 40 min. These steps were repeated with reactions of 1 µM LS with 1 mM L-cysteine and 1 mM D-cysteine, 1 µM LS with 5 mM OAS and either 1 mM D-cysteine or D-cysteine methyl ester, and 1 µM S224A with 5 mM OAS and 1 mM L-cysteine methyl ester. 4.2.8 Stopped-flow spectroscopy Pre-steady-state kinetic analysis of the reaction between LS, OAS, and L-cysteine or D-cysteine was performed on a model SF-61DX2 stopped-flow spectrophotometer from TgK Scientific. The stopped-flow reactions were performed in 50 mM HEPES-NaOH pH 7.5 at 25 °C. A solution of the enzyme (20 µM or 60 µM) was mixed with a fixed concentration of OAS (5 mM or 10 mM) and varying concentrations of L-cysteine or D-cysteine (0.2-5 mM) under the pseudo-first-order regime. The time-dependent absorbance changes in the PLP absorbance spectra were recorded in 107 triplicate at a single wavelength (428 nm or 460 nm) using a photomultiplier, and at multiple wavelengths (300−700 nm) using a photodiode array detector. To extract observed rate constants, a double exponential equation was fitted to the averaged data from the single-wavelength stopped-flow experiments using Kinetic Studio (TgK Scientific). If the observed rate constant showed a hyperbolic dependence on the concentration of the amino acid, then the hyperbolic function indicated in Equation 4.1 was fitted to the data using a non-linear least squares regression analysis in the computer program Origin (version 8.5). Equation 4.1 𝑘&'( = )!![+]-".[+] where kobs is the observed rate constant, Kd is the dissociation constant, [L] is the concentration of the amino acid substrate or product, and kaa is the limiting rate constant for the formation of the external aldimine or the decay of the a-aminoacrylate intermediate. If the observed rate constant had a linear dependence on the concentration of the amino acid, then the bimolecular rate constant (kon) was determined from the slope of a linear fit using a linear regression analysis in the computer program Origin (version 8.5). The dissociation rate constant (koff) was calculated by multiplying the kon and Kd values as indicated in Equation 2.3. 4.2.9 Absorbance assays with potential inhibitors of lanthionine synthase Absorbance spectra were recorded from 300-600 nm using a PerkinElmer Lambda 25 UV-visible spectrophotometer. Spectra of a 1 mL solution containing 10 µM LS in 100 mM HEPES-NaOH pH 7.4 were recorded at 25 °C. Spectral changes were observed after further addition of 10 mM of each inhibitor (D,L-2-aminopimelic acid, D,L-2-aminosuberic acid, S-(2-aminoethyl)-L-cysteine, S-(2-boronoethyl)-L-cysteine, S-(2-carboxyethyl)-L-cysteine, S-allyl-L-cysteine, L,L-2,6-diaminopimelic acid, meso-2,6-diaminopimelic acid, and meso-oxa-2,6-diaminopimelic acid). 108 4.2.10 Fluorescence spectroscopy Fluorescence spectra of LS with inhibitors were recorded on a PerkinElmer LS45 fluorescence spectrometer at 25 °C using a PerkinElmer PTP-1 Peltier temperature programmer. A 3 mL solution of LS (1-2 μM) was mixed with increasing concentrations of inhibitors in 100 mM HEPES-NaOH pH 7.4. The excitation wavelength was set to the absorbance maxima of either the external aldimine or a-aminoacrylate species that formed in the presence of the inhibitor. The fluorescence spectra were recorded in triplicate from 480-650 nm following a 2 min incubation time. The excitation and emission slits were set to 10 nm. Inhibitor binding constants (Kd) were determined by fitting a quadratic binding isotherm (Equation 4.2) to the change in fluorescence using a non-linear least squares regression analysis in the computer program Origin (version 8.5). Equation 4.2 ∆F = -∆F"#$2E2 0 1E2 + L2 + 𝐾/ − [(E2 + L2 + 𝐾/)3 − 4E2L2]4/37 where DF is the change in fluorescence emission, DFmax is the maximum change in fluorescence emission, E0 is the total enzyme concentration, L0 is the total inhibitor concentration, and Kd is the equilibrium dissociation constant for the external aldimine or a-aminoacrylate species of LS. 4.2.11 Steady-state kinetic inhibition assays The steady-state rate of H2S formation by LS in the presence of L-cysteine and an inhibitor (L,L-DAP or meso-DAP) was monitored by a continuous spectrophotometric assay that follows the reaction of H2S with lead acetate to form lead sulfide.132 The kinetic reactions were performed in triplicate at 25 °C in a total volume of 1 mL in 100 mM HEPES-NaOH pH 7.4 with 0.4 mM lead acetate. The concentration of L-cysteine was varied from 0.6-25 mM, and the concentration of the inhibitor was fixed from 0-400 μM. The reactions were initiated with the addition of 32 nM of LS. The initial velocity of the reaction was determined by the change in absorbance at 390 nm over time using an extinction coefficient of 5500 M-1 cm-1. The rate equation for competitive inhibition (Equation 4.3) was then fitted to the initial velocity data as a function of substrate and inhibitor concentration using a non-linear least squares regression analysis in the computer program Origin (version 8.5) to determine the inhibition constant (Ki) for the inhibitor. 109 Equation 4.3 v! = V"#$[S]𝐾6 -1 + [I]𝐾!0 + [S] where vi is the initial velocity, Vmax is the maximal velocity, [S] is the amino acid substrate concentration (L-cysteine), KM is the Michaelis constant for L-cysteine, [I] is the inhibitor concentration, and Ki is the inhibition constant for the inhibitor. 4.2.12 Substrate docking simulations Molecular docking simulations were carried out using AutoDock VINA (version 1.2.0). The starting coordinates of LS complexed with L,L-lanthionine (PDB entry 5XEM at 1.84 Å resolution) were obtained from the Protein Data Bank (PDB).82 The lanthionyl-PLP complex coordinates were removed from the original PDB file. Input files for the PLP-substrate adducts were generated with hydrogen atoms using ZINC20.167 AutoDock Tools was used to prepare the ligand and protein input files for docking simulations. Hydrogen atoms were added to the protein, and Gasteiger partial atomic charges were assigned to both the ligand and protein. The search space was confined to a 30 Å ´ 30 Å ´ 30 Å box centred at 25, 50, and 48 to include the site of the original PLP cofactor. Default parameters were used, and the number of energy evaluations was set to 250000. Solution structures were clustered according to their conformational similarities and then ranked from lowest to highest energy within each cluster. 4.3 Results 4.3.1 Properties of purified Fn1732 The putative lanthionine epimerase fused with a C-terminal His6-tag was overexpressed in E. coli and purified using Ni-NTA affinity chromatography. To confirm the purity and approximate size of the protein, SDS-PAGE was performed. The protein appeared as one band with an apparent molecular mass of ~33 kDa (Figure 4.1). This result is comparable to the molecular mass calculated from the amino acid sequence (33 239 Da). The absorbance spectrum of the protein showed a major peak at 280 nm, indicating the absence of any additional cofactors or metals. 110 Figure 4.1 SDS-PAGE gel of purified Fn1732. SDS 12% polyacrylamide gel showing Fn1732 after purification. Lane 1, PageRuler pre-stained protein ladder; Lane 2, purified Fn1732 after elution from the Ni-NTA column. 4.3.2 UV-visible spectroscopy The UV-visible absorbance spectrum of LS showed a major peak at 408 nm and a minor peak at 320 nm, which is attributed to the ketoenamine tautomer of the internal aldimine form of the PLP cofactor (Figure 4.2 (A)). The addition of OAS resulted in a shift of the major PLP absorbance band to 460 nm, concomitant with a significant increase at 320 nm. This spectral shift denotes formation of the a-aminoacrylate intermediate following b-elimination of the acetate group from the external aldimine with OAS. Subsequent addition of D-cysteine caused a shift from 460 nm to 428 nm and a decrease at 320 nm due to formation of the external aldimine with meso-lanthionine. Likewise, D-cysteine methyl ester readily reacted with the a-aminoacrylate to form the external aldimine with D,L-lanthionine methyl ester, as evidenced by a shift in λmax to 428 nm. In contrast, the addition of L-cysteine methyl ester to the a-aminoacrylate intermediate did not result in a change in the absorbance spectra, indicating that it is unable to dock into the second substrate binding site and react with the a-aminoacrylate. In wild-type LS, the b-hydroxyl group of S224 engages in hydrogen bond contact with the carboxylate group of lanthionine, stabilizing the formation of the product. We speculated that this residue prevents the binding of L-cysteine methyl 180 kDa130 kDa100 kDa70 kDa55 kDa40 kDa35 kDa25 kDaLane 1 Lane 2 111 ester. Therefore, we tested the reaction of OAS and L-cysteine methyl ester with the S224A variant. The absorbance spectrum of S224A in the resting state was similar to wild-type LS in that it had major and minor peaks at 405 nm and 320 nm, respectively (Figure 4.2 (B)). The a-aminoacrylate intermediate was formed after OAS was added, as shown by the lmax at 460 nm. The addition of L-cysteine methyl ester induced a shift in the PLP absorbance maximum to 410 nm with a shoulder at 485 nm, indicating formation of the external aldimine with L,L-lanthionine methyl ester. Figure 4.2 Absorbance spectra of wild-type lanthionine synthase and S224A with OAS and cysteine analogs. UV-visible absorbance spectra of (A) 50 μM wild-type LS and (B) 10 μM S224A in the internal aldimine form (black line) and after mixing with 1 mM OAS (black dashed line). Spectral changes were observed after further addition of 1 mM D-cysteine (grey line), D-cysteine methyl ester (grey dashed line), or L-cysteine methyl ester (grey dotted line). All 1 mL reactions were performed in 100 mM HEPES-NaOH pH 7.4 at 25 °C. 4.3.3 Steady-state kinetic analysis of lanthionine production Diaminopimelic acid dehydrogenase (DAPDH) catalyzes the NADP-dependent oxidative deamination reaction of meso-2,6-diaminopimelic acid (meso-DAP) to L-2-amino-6-ketopimeliate and ammonia. Given that meso-lanthionine is an analog of meso-DAP, we measured the steady-state kinetic parameters associated with meso-lanthionine or D,L-lanthionine methyl ester formation in a coupled spectrophotometric assay with LS, DAPDH, OAS, and D-cysteine or D-cysteine methyl ester by following NADPH formation at 340 nm. As shown in Figure 4.3 (A) and (B), the initial velocity increased hyperbolically as a function of D-cysteine or D-cysteine methyl ester A B 112 concentration. The D-cysteine analogs exhibited comparable kcat, KM, and kcat/KM values of 0.081 ± 0.002 s-1, 0.16 ± 0.01 mM, and 500 ± 39 s-1 M-1 for D-cysteine, and 0.078 ± 0.003 s-1, 0.23 ± 0.02 mM, and 340 ± 43 s-1 M-1 for D-cysteine methyl ester, respectively. There was no activity with S224A when L-cysteine methyl ester was substituted for the D-cysteine analogs, reaffirming that the D,L-diastereomer of the products was being formed. The same coupled assay was performed with the putative lanthionine epimerase (Fn1732) with L,L-lanthionine to test its activity and confirm the production of meso-lanthionine. L,L-lanthionine is not commercially available, so it was synthesized from a reaction of LS with L-cysteine and isolated through centrifugation using a centricon with a 30 kDa MWCO filter. A Michaelis-Menten plot was generated of the initial velocity versus increasing concentrations of L,L-lanthionine (Figure 4.3 (C)). The kcat, KM, and kcat/KM values were determined to be 0.070 ± 0.001 s-1, 1.8 ± 0.1 mM, and 39 ± 3 s-1 M-1, respectively. All the kinetic parameters determined from the coupled assay with DAPDH are summarized in Table 4.1. Table 4.1 Steady-state kinetic parameters associated with meso-lanthionine or D,L-lanthionine methyl ester production by lanthionine synthase in the presence of OAS and D-cysteine or D-cysteine methyl ester, respectively, and meso-lanthionine production by Fn1732 in the presence of L,L-lanthionine.Substrate kcat (s-1) KM (mM) kcat/KM (s-1 M-1) D-cysteine 0.081 ± 0.002 0.16 ± 0.01 500 ± 39 D-cysteine methyl ester 0.078 ± 0.003 0.23 ± 0.02 340 ± 43 L,L-lanthionine 0.070 ± 0.001 1.8 ± 0.1 39 ± 3 113 Figure 4.3 Determination of steady-state kinetic parameters associated with lanthionine production for lanthionine synthase and Fn1732. A plot of the initial velocity versus concentration was generated for the reactions of LS with OAS and (A) D-cysteine or (B) D-cysteine methyl ester to produce meso-lanthionine and D,L-lanthionine methyl ester, respectively, and (C) the reaction of Fn1732 with L,L-lanthionine to form meso-lanthionine. 4.3.4 HPLC analysis of lanthionine products The ability of wild-type LS and S224A to produce lanthionine analogs was analyzed using reversed-phase HPLC. Following a 2 h incubation of LS with OAS and D-cysteine, the HPLC chromatogram revealed the appearance of a peak that eluted with the same retention time as a lanthionine standard (Figure 4.4 (A)). There were also negligible peaks associated with OAS and D-cysteine starting material. As shown in Figure 4.4 (B), incubation of OAS and D-cysteine methyl ester with LS generated a major peak associated with lanthionine formation and minor peaks associated with OAS and D-cysteine methyl ester. The reaction of S224A with OAS and L-cysteine methyl ester generated similar results with major and minor peaks associated with lanthionine formation and leftover starting material, respectively (Figure 4.4 (C)). These data show that LS is capable of catalyzing reactions with different cysteine analogs to produce different forms of lanthionine (refer to Figure 1.15 for reaction products). A B C 114 Figure 4.4 HPLC analysis of lanthionine analogs produced by lanthionine synthase. Dansylated products obtained following a 2 h incubation of 1 μM LS with 5 mM OAS and 1 mM (A) D-cysteine or (B) D-cysteine methyl ester, and (C) 1 μM S224A with 5 mM OAS and 1 mM L-cysteine methyl ester (top panels). The middle panels show 1 mM of a pure lanthionine standard (L,L- and meso- mixture), and the bottom panels show the pure standards of 5 mM OAS and 1 mM D-cysteine, D-cysteine methyl ester, and L-cysteine methyl ester. Given that D-cysteine was able to dock into the second substrate binding site in the reaction with LS and OAS, we wanted to determine if LS could form meso-lanthionine in the presence of equimolar amounts of L-cysteine and D-cysteine (Figure 4.5). Figure 4.5 Lanthionine synthase reaction of L-cysteine and D-cysteine to form L,L-lanthionine, meso-lanthionine, and H2S. In the reaction of LS with L-cysteine, L,L-lanthionine is formed when a second equivalent of L-cysteine reacts with the a-aminoacrylate intermediate (refer to Figure 1.11). This was confirmed through HPLC analysis when the addition of L-cysteine to LS generated a single peak in the HSCOO-NH2HSNH2+ SCOO-NH2-OOCNH2L-cysteine D-cysteine L,L-lanthionine+ SCOO-NH2-OOCNH2meso-lanthionine+ H2SCOO- LSLS + OAS + D-cysteine LS + OAS + D-cysteine methyl ester S224A + OAS + L-cysteine methyl ester A B C 115 chromatogram that had the same retention time as the first of two peaks of a lanthionine standard (L,L- and meso- mixture) (Figure 4.6 (A)). The first peak of the lanthionine standard corresponds to the L,L-isomer, and the second peak corresponds to the meso-isomer. When LS was reacted with equimolar amounts of L-cysteine and D-cysteine, it resulted in the appearance of two peaks that corresponded to the lanthionine standard (Figure 4.6 (B)). Notably, meso-lanthionine was produced in favour of L,L-lanthionine with a 3:1 ratio, as determined from the area of the two peaks. Figure 4.6 HPLC analysis of L,L-lanthionine and meso-lanthionine production by lanthionine synthase. Products obtained from reactions of 1 μM LS with (A) 1 mM L-cysteine and (B) 1 mM L-cysteine and D-cysteine are shown on the top panels. Pure standards of 1 mM lanthionine (L,L- and meso- mixture), L-cysteine, and D-cysteine are shown on the middle and bottom panels, respectively. 4.3.5 Pre-steady-state kinetic analysis with OAS and L-cysteine or D-cysteine L,L-lanthionine or meso-lanthionine formation occurs when L-cysteine or D-cysteine binds at the second substrate binding site and attacks the a-aminoacrylate intermediate. To measure the rates and binding constants associated with the reaction of L-cysteine or D-cysteine with the a-aminoacrylate intermediate, stopped-flow experiments were performed. The a-aminoacrylate LS + L-cysteine LS + L-cysteine + D-cysteine B A 116 intermediate was initially formed by incubating LS with OAS. Within 2 s, rapid mixing with L-cysteine resulted in the decay of the a-aminoacrylate intermediate, as indicated by a decrease at 460 nm (Figure 4.7 (A)). From 2-42 s, this was followed by the formation of the external aldimine with L,L-lanthionine, signified by an increase in absorbance at 428 nm. The reaction of the a-aminoacrylate species with D-cysteine produced the same result; from 0.001-21 s, there was a decrease in lmax at 460 nm and an increase in lmax at 428 nm, indicating the conversion of the a-aminoacrylate species into the external aldimine with meso-lanthionine (Figure 4.7 (B)). Between the two enantiomers, D-cysteine reacted with the a-aminoacrylate intermediate faster than L-cysteine as shown by the greater loss of absorbance lmax at 460 nm following rapid mixing. Figure 4.7 Stopped-flow spectra of lanthionine synthase with OAS and L-cysteine or D-cysteine. Time-resolved absorbance spectra of 60 µM LS pre-mixed with 10 mM OAS (black line) and following the reaction with 5 mM (A) L-cysteine or (B) D-cysteine (grey lines). Spectra were acquired on a log time base from (A) 0.001-2 s (top) and 2-42 s (bottom), and (B) 0.001-2 (top) and 2-21 s (bottom) for the reactions with L-cysteine and D-cysteine, respectively. The arrows represent the direction of changes in absorbance over time. A B 117 The reaction of LS with OAS and L-cysteine or D-cysteine undergoes conversion from the a-aminoacrylate intermediate to the external aldimine with an intermediary Michaelis complex. (Figure 4.8). Single-wavelength stopped-flow experiments at 428 nm and 460 nm were performed to measure the rates and binding affinities associated with the formation and decay of these PLP intermediates. For these experiments, LS was initially reacted with OAS to form the a-aminoacrylate intermediate. This species was then mixed with increasing L-cysteine or D-cysteine concentrations. The parameters associated with these kinetic events are summarized in Table 4.2. Figure 4.8 Intermediates involved in the reaction of cysteine with the a-aminoacrylate intermediate. The rate constants kon and koff are associated with the formation of the Michaelis complex between cysteine and the a-aminoacrylate intermediate. The rate constant kaa represents the rate of nucleophilic attack of the cysteine thiolate on the a-aminoacrylate intermediate following formation of the Michaelis complex. Table 4.2 Pre-steady-state kinetic parameters for the reactions of lanthionine synthase with OAS and L-cysteine or D-cysteine at 460 nm and 428 nm.Wavelength Substrate kon (mM-1 s-1) koff (s-1) Kd (mM) kaa (s-1) 460 nm L-cysteine 49 ± 3 56 ± 12 1.1 ± 0.2 4.2 ± 0.4 D-cysteine 130 ± 2 44 ± 6 0.33 ± 0.04 13 ± 1 428 nm L-cysteine 58 ± 3 43 ± 3 0.74 ± 0.02 0.097 ± 0.001 D-cysteine 27 ± 2 11 ± 1 0.39 ± 0.02 0.306 ± 0.005 PLPNH+COO-Lys42NH3+NH2-OOCS-konkoffPLPNH+COO-Lys42NH2+NH2-OOCS-HkaaPLPNH+COO-S-OOCNH2α-aminoacrylate Michaelis complex external aldimineLys42NH2 118 For the reaction of LS with OAS and L-cysteine, a double exponential equation was fitted to the biphasic absorbance traces at 460 nm (Figure 4.9 (A)). As shown in Figure 4.9 (B), the observed rate constant for the initial fast phase (kobs1) displayed a linear dependence on L-cysteine concentration. The bimolecular rate constant, determined from the slope of a linear fit of the data, was 49 ± 3 mM-1 s-1. The observed rate constant for the slower phase (kobs2) showed a hyperbolic dependence on L-cysteine concentration. A fit of Equation 4.1 to the data gave Kd and kaa values of 1.1 ± 0.2 mM and 4.2 ± 0.4 s-1, respectively. The kaa represents the rate constant associated with the nucleophilic attack of the thiolate group of L-cysteine on the a-aminoacrylate intermediate. At 428 nm, the absorbance traces were also biphasic (Figure 4.9 (C)). A fit of a double exponential equation to the traces resulted in a linear dependence on L-cysteine concentration for the fast phase (kobs1), yielding a bimolecular rate constant of 58 ± 3 mM-1 s-1. The slower observed rate constant (kobs2) showed a hyperbolic dependence on L-cysteine concentration, giving a Kd of 0.74 ± 0.02 mM and a kaa of 0.097 ± 0.001 s-1 (Figure 4.9 (D)). 119 Figure 4.9 Reaction of lanthionine synthase with OAS and increasing concentrations of L-cysteine. Single-wavelength absorbance traces at (A) 460 nm and (C) 428 nm following rapid mixing of 20 µM LS and 5 mM OAS with L-cysteine (0.2-4 mM). The arrows indicate the direction of the changes in absorbance with increasing amounts of L-cysteine. Linear dependence of kobs1 (inset) and hyperbolic dependence of kobs2 on L-cysteine concentration measured at (B) 460 nm and (D) 428 nm. Similarly, the absorbance traces at 460 nm and 428 nm were biphasic when D-cysteine was substituted for L-cysteine in the reaction with LS and OAS (Figure 4.10 (A, C)). A double exponential equation was fitted to each kinetic phase to extract slow (kobs1) and fast (kobs2) observed rate constants. A plot of kobs1 versus D-cysteine concentration showed a linear dependence at both wavelengths, producing kon rates of 130 ± 2 mM-1 s-1 (460 nm) and 27 ± 2 mM-1 s-1 (428 nm). The second observed rate constant (kobs2) was hyperbolically dependent on D-cysteine concentration, yielding Kd and kaa values of 0.33 ± 0.04 mM and 13 ± 1 s-1 at 460 nm, and 0.39 ± 0.02 mM and 0.306 ± 0.005 s-1 at 428 nm, respectively (Figure 4.10 (B, D)). A comparison of the Kd and kaa A B C D 120 values obtained for L-cysteine and D-cysteine presented in Table 4.2 reveals that D-cysteine exhibits a 2-3-fold tighter binding affinity for the second substrate binding pocket and reacted 3 times faster with the a-aminoacrylate intermediate compared to L-cysteine. Figure 4.10 Reaction of lanthionine synthase with OAS and increasing concentrations of D-cysteine. Absorbance traces at (A) 460 nm and (C) 428 nm following rapid mixing of 20 µM LS and 5 mM OAS with D-cysteine (0.2-4 mM). The arrows indicate the direction of the changes in absorbance with increasing amounts of D-cysteine. Linear dependence of kobs1 (inset) and hyperbolic dependence of kobs2 on D-cysteine concentration measured at (B) 460 nm and (D) 428 nm. 4.3.6 Spectral analysis of potential inhibitors Nine analogs of diaminopimelic acid and cysteine were tested to see if they could act as potential inhibitors of LS (refer to Table 4.4 for a list of the inhibitors and their respective structures). Changes in the UV-visible absorbance spectra were followed to determine if the analogs could A B C D 121 form an imine linkage with the PLP cofactor. As shown in Figure 4.11, the addition of D,L-2-aminopimelic acid, D,L-2-aminosuberic acid, S-(2-boronoethyl)-L-cysteine, S-(2-carboxyethyl)-L-cysteine, S-allyl-L-cysteine, L,L-2,6-diaminopimelic acid, or meso-2,6-diaminopimelic acid to LS resulted in a shift in absorbance from 406 nm to 411-450 nm, indicating that the enzyme forms the external aldimine with the inhibitor. In contrast, addition of S-(2-aminoethyl)-L-cysteine or meso-oxa-2,6-diaminopimelic acid to LS resulted in the formation of the a-aminoacrylate intermediate, as evidenced by absorbance shifts to 461 nm and 462 nm, respectively. Thus, these two substrates undergo a β-elimination reaction following the formation of the external aldimine. The lmax values determined from the reactions of LS and each inhibitor are summarized in Table 4.3. Table 4.3 Absorbance maxima (lmax) determined from the absorbance spectra of lanthionine synthase with potential inhibitors.Inhibitor lmax (nm) D,L-2-aminopimelic acid 415 D,L-2-aminosuberic acid 411 S-(2-aminoethyl)-L-cysteine 461 S-(2-boronoethyl)-L-cysteine 424 S-(2-carboxyethyl)-L-cysteine 424 S-allyl-L-cysteine 450 L,L-2,6-diaminopimelic acid 431 meso-2,6-diaminopimelic acid 430 meso-oxa-2,6-diaminopimelic acid 462 122 Figure 4.11 Absorbance spectra of lanthionine synthase with potential inhibitors. UV-visible spectra from 300-600 nm of 10 µM LS in the internal aldimine form (black line) and following a 5 min incubation with 10 mM (A) D,L-2-aminopimelic acid, (B) D,L-2-aminosuberic acid, (C) S-(2-aminoethyl)-L-cysteine, (D) S-(2-boronoethyl)-L-cysteine, (E) S-(2-carboxyethyl)-L-cysteine, (F) S-allyl-L-cysteine, (G) L,L-2,6-diaminopimelic acid, (H) meso-2,6-diaminopimelic acid, and (I) meso-oxa-2,6-diaminopimelic acid (grey lines). All spectra were recorded in 1 mL 100 mM HEPES-NaOH pH 7.4 at 25 °C. 4.3.7 Fluorescence inhibition analysis Fluorescence titration experiments were also performed to determine the relative binding affinities of each inhibitor. The PLP cofactor was excited at the absorbance lmax following the addition of inhibitor to the enzyme (refer to Table 4.3). The change in the maximum fluorescence was plotted A B C D E F G H I 123 versus the inhibitor concentration and Equation 4.2 was fitted to the data (Figure 4.12). The inhibitors tested exhibited Kd values ranging from 0.5-120 µM, with S-(2-boronoethyl)-L-cysteine exhibiting the tightest binding affinity (Table 4.4). Notably, L,L-2,6-diaminopimelic acid binds to LS with a 12-fold weaker binding affinity compared to its diastereomer, meso-2,6-diaminopimelic acid. Table 4.4 Structures and experimentally determined Kd values for potential inhibitors of LS.Inhibitor Structure Kd (µM) D,L-2-aminopimelic acid 4.5 ± 0.1 D,L-2-aminosuberic acid 120 ± 9 S-(2-aminoethyl)-L-cysteine 70 ± 2 S-(2-boronoethyl)-L-cysteine 0.5 ± 0.1 S-(2-carboxyethyl)-L-cysteine 6.1 ± 0.5 S-allyl-L-cysteine 30 ± 2 L,L-2,6-diaminopimelic acid 29 ± 2 meso-2,6-diaminopimelic acid 2.5 ± 0.2 meso-oxa-2,6-diaminopimelic acid 0.7 ± 0.1 124 Figure 4.12 Fluorescence emission spectra of lanthionine synthase with potential inhibitors. A plot of the change in PLP fluorescence emission maxima versus inhibitor concentration was generated for the reactions of 1-2 µM LS with increasing concentrations of (A) D,L-2-aminopimelic acid, (B) D,L-2-aminosuberic acid, (C) S-(2-aminoethyl)-L-cysteine, (D) S-(2-boronoethyl)-L-cysteine, (E) S-(2-carboxyethyl)-L-cysteine, (F) S-allyl-L-cysteine, (G) L,L-2,6-diaminopimelic acid, (H) meso-2,6-diaminopimelic acid, and (I) meso-oxa-2,6-diaminopimelic acid (grey lines). 4.3.8 Steady-state kinetic inhibition analysis The ability of meso-DAP and L,L-DAP to inhibit LS activity was tested using the lead acetate steady-state assay. The initial velocity of the reaction was measured using varying L-cysteine concentrations and fixed inhibitor concentrations. Double reciprocal plots were generated from the initial velocity of the reaction versus L-cysteine concentration (Figure 4.13). The competitive A B C D E F G H I 125 inhibition equation (Equation 4.3) was fitted to the data, which gave inhibition constants (Ki) of 198 ± 45 µM for L,L-DAP and 75 ± 16 µM for meso-DAP (Table 4.5). From the interpretation of the fluorescence and kinetic results, it was concluded that meso-DAP may act as a more potent inhibitor compared to L,L-DAP, as evidenced by lower Kd and Ki values. Table 4.5 Inhibition constants of L,L-DAP and meso-DAP for lanthionine synthase.Inhibitor Ki (µM) L,L-2,6-diaminopimelic acid 198 ± 45 meso-2,6-diaminopimelic acid 75 ± 16 Figure 4.13 Inhibition of lanthionine synthase by L,L-DAP and meso-DAP. Double reciprocal plots of the initial velocity versus L-cysteine concentration for the reactions with LS and varying amounts of (A) L,L-DAP or (B) meso-DAP. 4.3.9 Substrate docking simulations To further investigate L,L-DAP and meso-DAP as LS inhibitors, a molecular model of each inhibitor in the LS active site was generated using AutoDock VINA (version 1.2.0). Lanthionine synthase complexed with L,L-lanthionine (PDB entry 5XEM at 1.84 Å resolution) was used as the starting coordinates of the protein.82 The lanthionyl-PLP complex was removed from the original PDB file to allow for docking of the PLP-DAP adduct into the active site. For a structure to be considered a viable fit, the PLP portion of the ligand had to overlay with the PLP cofactor in the A B 126 original protein file. A model of L,L-DAP into the LS active site reveals that the a-carboxylate groups of the inhibitor engages in hydrogen bond contact with the backbone NH of S70 and the b-hydroxyl group of S224, and the terminal amino group interacts with the carbonyl groups of G222 and G220 (Figure 4.14 (A)). In the meso-DAP model, the same hydrogen bonding still occurs with S70, S224, and G220, but there is no interaction with G222 (Figure 4.14 (B)). However, there is a new hydrogen bond formed between the terminal amino group of the product and the b-hydroxyl group of S70, which may account for the tighter binding of the D-diastereomer. Figure 4.14 Molecular model of DAP analogs in lanthionine synthase. Model of (A) L,L-DAP and (B) meso-DAP into the active site of LS. The PLP-DAP complexes (magenta) are overlayed with the PLP-lanthionyl complex present in PDB entry 5XEM (resolution at 1.84 Å).82 The carbon atoms of LS active site residues are coloured grey, the carbon atoms of the PLP cofactors are coloured green, and the carbon atoms of lanthionyl are coloured cyan. Hydrogen bonds between the DAP-PLP complexes and active site residues in LS are shown as black dashed lines. 4.4 Discussion 4.4.1 Cysteine derivatives can react with LS to form different lanthionine products LS is part of the fold type II PLP-dependent enzyme family that also includes cystathionine b-synthase (CBS) and O-acetylserine sulfhydrylase (OASS). These enzymes catalyze b-replacement reactions through an a-aminoacrylate intermediate and exhibit a degree of substrate promiscuity. S224 S224 G222 G222 G220 G220 S70 A B S70 127 Although LS has a catalytic preference for lanthionine biosynthesis, it exhibits substrate promiscuity by being able to catalyze the reaction of OAS and sulfide to form L-cysteine and acetate.44 In the absence of sulfide, the reaction leads to the accumulation of the a-aminoacrylate species. This allows for nucleophilic attack from an additional substrate binding into the active site. We were interested to see if the slightly relaxed substrate scope of LS for the second substrate binding pocket could be utilized to synthesize different lanthionine products using D-cysteine, D-cysteine methyl ester, and L-cysteine methyl ester as substrates. UV-visible absorbance spectra showed that the addition of D-cysteine or D-cysteine methyl ester caused a spectral shift in absorbance that likely indicated the formation of the external aldimine with the respective lanthionine product. In the reaction with L-cysteine methyl ester, the external aldimine was only formed when reacted with the S224A variant, suggesting that the b-hydroxyl group of S224 prevented L-cysteine methyl ester from binding to the wild-type enzyme. 4.4.2 Lanthionine analogs for use in peptide synthesis When presented with challenging environmental conditions, bacteria can respond and adapt to these changes by remodeling the structure and composition of their peptidoglycan (PG).168 During bacterial growth, PG remodeling results in the release of PG fragments into the surrounding environment. While some of these fragments are recycled for cell wall biosynthesis, they can also be used as messenger molecules to modulate a response from neighbouring bacteria or a host organism.169 PG fragments are examples of pathogen-associated molecular patterns (PAMPs), molecules that can be recognized by specialized receptors to help distinguish between pathogens and host cells.170 In humans, nucleotide binding and oligomerization domain proteins (NODs) are receptors that are implicated in the intracellular detection of PAMPs. NOD1 and NOD2 both sense peptidoglycan through the detection of different stem peptide fragments. NOD2 senses the L-Ala-D-Glu dipeptide found in most PGs.171 This conserved peptidoglycan motif sensed by NOD2 allows it to act as a general sensor of PG degradation products of both gram-negative and gram-positive bacteria. Conversely, NOD1 mediates the recognition of the tripeptide L-Ala-D-Glu-meso-DAP, limiting the receptor to gram-negative bacteria due to the additional requirement of meso-DAP.172 Due to their ability to modify the structure of their peptide stem, a few pathogenic and commensal bacteria have been found to evade the NOD1 response through amidation of meso- 128 DAP.173,174 Interestingly, it was found that a tripeptide containing meso-lanthionine in place of meso-DAP was detected by NOD1.173 Although the structural difference between meso-lanthionine and meso-DAP may be subtle, it may still have a significant impact on how PG fragments are sensed or metabolized. Therefore, the production of meso-lanthionine and its derivatives by LS could be useful in the development of synthetic PG fragments to expand our knowledge of PG biosynthesis, remodeling, and sensing. 4.4.3 LS can produce meso-lanthionine in the presence of D-cysteine F. nucleatum is unique compared to other gram-negative bacteria in that instead of meso-DAP, meso-lanthionine takes place in the third position of the peptide crosslink within the cell wall. If the reaction of LS with L-cysteine produces L,L-lanthionine, then how is F. nucleatum producing meso-lanthionine for incorporation into its peptidoglycan layer? Since the lanthionine analog experiments showed that LS can accommodate docking D-cysteine into the second substrate binding site, we reacted LS with equimolar amounts of L-cysteine and D-cysteine to determine which lanthionine diastereomer would be favourably produced. HPLC analysis confirmed that meso-lanthionine was produced preferentially in a 3:1 ratio to L,L-lanthionine. The preference for the meso-isomer may be attributed to D-cysteine binding tighter to the second binding site and reacting faster with the a-aminoacrylate intermediate when compared to L-cysteine. Although we demonstrated that the reaction of LS with D-cysteine can occur, it is still a question of if it is physiologically relevant. 4.4.4 Availability of D-cysteine D-amino acids, once considered non-functional and not naturally present in living organisms, are essential for microbial physiology.175 In particular, D-glutamic acid and D-alanine are key components that make up the peptidoglycan layer in most bacteria. The generation of D-amino acids occurs mainly via racemization, whether it be spontaneously or through a racemase. Cysteine has a high propensity for spontaneous racemization because it is the fastest amino acid to do so with a half-life (t1/2) of 2 days.176 It is also possible that D-cysteine can be produced from L-cysteine enzymatically through a PLP-dependent serine racemase, which is encoded in the F. nucleatum genome.177 The presence of a serine racemase in the genome may be taken as an indication of the 129 abundance of D-cysteine in the environment, but there is a limited amount of information to confirm its availability. Nonetheless, a recent study found that D-cysteine is greatly enriched in embryonic mouse brains with levels as high as 4.5 mM.178 In adult mice, D-cysteine is present in the brain in lower amounts (50 µM), but it has also been found in the eye (150 µM) and the pancreas (40 µM).179 The substantial decline in D-cysteine levels in mice over their lifespan was attributed to D-cysteine inhibiting neural progenitor cell proliferation, thus acting as a negative regulator of growth factor signaling during development of the brain.178 Additional indirect evidence of D-cysteine availability in the environment is provided by the presence of a D-cysteine desulfhydrase in specific species of plants and bacteria. D-cysteine desulfhydrase is a PLP-dependent enzyme that facilitates the a,b-elimination reaction of D-cysteine to form H2S, pyruvate, and NH3.180,181 In plants, H2S is important for various development processes and stress responses. In Arabidopsis, H2S produced by D-cysteine desulfhydrase protects the plant against biotic and abiotic stresses through upregulation of multiple stress-related genes and inhibition of accumulation of reactive oxygen species (ROS).182 D-cysteine desulfhydrase-mediated H2S production also has a role in the plant stress response to cadmium, a toxic element that negatively impacts plant growth and development.183 In E. coli, D-cysteine desulfhydrase facilitates growth by utilizing H2S as a sulfur source. Conversely, the absence of this enzyme inhibits growth due to D-cysteine acting as an inhibitor of threonine deaminase, a key enzyme involved in the isoleucine biosynthesis pathway.184 In regards to Fusobacteria species, it was found that Fusobacterium sulci and Fusobacterium varium exhibited high activity of D-cysteine desulfhydrase, while activity of this enzyme in F. nucleatum was absent.185 Coincidentally, the peptidoglycan structure of F. sulci and F. varium is based on diaminopimelic acid.186 F. nucleatum also does not encode a homolog for D-cysteine desulfhydrase, thus the consumption of D-cysteine by lanthionine synthase could possibly negate the need for this enzyme. 4.4.5 Fn1732 as a potential lanthionine epimerase In most gram-negative bacteria, meso-DAP is a precursor for L-lysine and an important component for cell wall biosynthesis.187 A key enzyme in the DAP pathway is DAP epimerase (DapF), which catalyzes the PLP-independent racemization reaction of L,L-DAP to meso-DAP. The reaction 130 proceeds without additional cofactors or metals because of two cysteine residues that act as a thiol and thiolate for general acid/base chemistry.93 These cysteine residues are conserved among other PLP-independent racemases such as histidine racemase (CntK) and O-ureidoserine racemase (DcsC).188,189 Given that lanthionine is a thioether analog to DAP, the characterization of an epimerase was considered as an alternative approach for determining the possible mechanism for F. nucleatum to produce meso-lanthionine. Although Fn1732 displays a low sequence identity compared to DapF (19.8%), the gene was considered a viable candidate for a lanthionine epimerase due to the presence of two cysteine residues (C67 and C209) that are conserved with the other amino acid epimerase and racemases mentioned above (Figure 4.15). Figure 4.15 Sequence alignment of Fn1732 with other PLP-independent epimerases/racemases. The primary sequence of Fn1732 from F. nucleatum with DAP epimerase, histidine racemase, and O-ureidoserine racemase was determined using Clustal MUSCLE.146 The protein sequences are as follows: Fn1732 (accession number: AAL93847.1), DapF, DAP epimerase from Escherichia coli (accession number: CAD6023061.1), CntK, histidine racemase from Staphylococcus aureus (accession number: BEV56855.1), and DcsC, O-ureidoserine racemase from Xanthomonas arboricola (accession number: CAE6712230.1). The conserved cysteine residues are shown in red. The activity of Fn1732 as a lanthionine epimerase was monitored through a coupled spectrophotometric assay. It was determined that Fn1732 could convert L,L-lanthionine to meso-lanthionine with a KM of 1.8 ± 0.1 mM and catalytic turnover of 0.070 ± 0.001 s-1. In comparison, DAP epimerase exhibits a 14-fold lower KM value (0.129 ± 0.02 mM) and an almost 2000-fold higher kcat value (120 ± 4 s-1) under similar experimental conditions.190 This suggests that L,L-lanthionine may not be the physiological substrate for Fn1732, and that this enzyme may act as a racemase on another amino acid. FN1732 60 ----QMMGGEFCGNASRAFASLLA ...... KEVGSGVYENSCASGTTALGYYL 220 DapF 62 IFNADGSEVAQCGNGARCFARFVR ...... RGAGE---TQACGSGACAAVAVG 228 CntK 65 ----VMSGNEFCGNATMSYIHHL- ...... PEIQSLIWENSCGSGTASIGVFN 222 DcsC 71 IWNSDGSAARQCGNGARCVAAWLV ...... RGVGE---TLACGSGACAAAVVL 238 131 4.4.6 Potential inhibitors of LS Lanthionine is an essential component of the cell wall of F. nucleatum, and therefore LS could be considered a drug target for controlling the growth of this bacterium. The starting point for potential inhibitors was to test analogs of cysteine and 2,6-diaminopimelic acid (DAP), a molecule with a similar in structure as lanthionine except that it lacks a thioether group. We established that all the tested cysteine and DAP analogs were able to form an imine linkage to the PLP cofactor of LS as either the external aldimine or a-aminoacrylate intermediate with Kd values ranging from 0.5-117 µM. Despite having a bulky boronic acid group, S-2-(boronoethyl)-L-cysteine exhibited the tightest binding affinity out of the inhibitors tested. D,L-2-aminosuberic acid had the weakest binding affinity, which could be attributed to having a longer chain that could prevent favourable interactions with amino acid residues in the active site. When a substrate binds to the active site, LS undergoes a conformational change that triggers closure of the active site. This process involves the movement of four loops: 69TSGNT73 (loop A), 94DTMS97 (loop B), 115DGSLGM120 (loop C), and 223MSIG226 (loop D). Loop A undergoes the most significant change when it is brought closer to the PLP cofactor through the substrate a-carboxylate forming a strong hydrogen bond network with the hydroxyl group of T69 and the backbone NH of S70 and T73 (Figure 4.16).44,82 This loop is conserved among OASS and CBS, resulting in similar substrate-induced conformational changes.79,191 The movement of loop A is also communicated to loop D, which plays an important role in product binding through hydrogen bond contact between the backbone NH and hydroxyl group of S224 to the terminal carboxylate group of the product. Differences in interactions with these key residues likely contribute to the different binding affinities of the tested inhibitors. 132 Figure 4.16 Hydrogen bond contacts of lanthionine synthase within the L,L-lanthionine external aldimine. A close-up of the active site in LS (PDB entry 5XEM at 1.84 Å resolution) shows the interactions between the PLP-lanthionyl complex and active site residues in loops A and D.82 The carbons of PLP cofactor are coloured green, lanthionyl is coloured cyan, and the amino acid residues in the active site are coloured grey. Hydrogen bonds are shown as black dashed lines. To better understand the influence of stereochemistry on interactions involved in inhibitor binding, docking simulations were performed with L,L-DAP and meso-DAP. A molecular model of meso-DAP in the LS active site revealed a new hydrogen bond that forms between the terminal amino group and the b-hydroxyl group on the side chain of S70. Notably, this residue also engages in hydrogen bonding with meso-oxa-DAP, except with the ether oxygen (Figure 4.17).82 This interaction may promote the a,b-elimination of D-serine to form the a-aminoacrylate species, as observed in the UV-visible experiments (refer to Figure 4.11 (I)). S224 S70 T69 T73 133 Figure 4.17 Molecular model of meso-oxa-DAP in the active site of lanthionine synthase. The PLP-bound meso-oxa-DAP adduct (magenta) is overlayed with the external aldimine of LS with L,L-lanthionine (PDB entry 5XEM at 1.84 Å resolution).82 The carbon atoms of the PLP cofactors are coloured green, the lanthionyl carbons are coloured cyan, and the carbons of LS active site residues are coloured grey. Hydrogen bonds are shown as black dashed lines. In order to observe the effects of stereochemistry on inhibition potential, kinetic assays were performed with L,L-DAP and meso-DAP in competition with L-cysteine. In conjunction with the fluorescence experiments, it was determined that LS is stereoselective towards meso-DAP compared to the L,L-diastereomer due to 3-fold and 12-fold lower Ki and Kd values, respectively. This result is similar to LS preferentially producing meso-lanthionine over L,L-lanthionine in the presence of both L-cysteine and D-cysteine. Given that both D-cysteine and meso-DAP also exhibit tighter binding affinity in comparison to their respective stereoisomers, perhaps we should consider the use of D-isomers for inhibitors of LS in future research. 4.5 Concluding Remarks The research presented herein provides a basis for the development of synthetic routes for lanthionine and lanthionine analogs. UV-visible absorbance studies and HPLC analysis showed that cysteine analogs D-cysteine, D-cysteine methyl ester, and L-cysteine methyl ester can add to the a-aminoacrylate intermediate of LS to form a lanthionine product. Isolation and S224 G222 G220 S70 134 characterization of these lanthionine analogs using nuclear magnetic resonance (NMR) spectroscopy could be used to establish certainty on the identity of these products. It was also found that LS can produce meso-lanthionine from the reaction with L-cysteine and D-cysteine. However, further research is required to accurately quantify D-cysteine levels within F. nucleatum to determine whether this reaction is physiologically relevant. It is likely that meso-lanthionine is produced through the activity of LS and not an epimerase due to the low lanthionine epimerase activity of Fn1732. The mechanism of Fn1732 could be further studied through site-directed mutagenesis of the conserved cysteine residues involved in catalysis. The racemization activity of this enzyme could also be tested on other amino acids to determine its physiological substrate. We also established that out of the nine tested inhibitors, meso-DAP may act as a potent inhibitor of LS because of a new hydrogen bond contact formed with S70. This result determined from molecular docking could be confirmed by generating a S70A variant of LS and assaying its activity with and without meso-DAP. Given that the only known biosynthetic route for F. nucleatum to produce lanthionine is through LS, inhibition of this enzyme may serve as a promising strategy in drug design to target the growth of this bacterium. 135 Chapter 5: Lysine 5,6-Aminomutase and KamBC 5.1 Chapter Summary Lysine 5,6-aminomutase (5,6-LAM) is a PLP- and AdoCbl-dependent enzyme involved in the lysine fermentation pathway of F. nucleatum. In the second step of this pathway, 5,6-LAM catalyzes the difficult 1,2-amino shift reaction of b-L-lysine to erythro-3,5-diaminohexanoate. During the isomerization reaction, the Co-C bond of the AdoCbl cofactor undergoes homolysis, generating cob(II)alamin and a 5′-deoxyadenosyl radical. Due to the highly reactive nature of the radical species generated through the reaction, 5,6-LAM undergoes irreversible oxidation of the AdoCbl cofactor to generate hydroxocobalamin, an inactive form of the enzyme. The genes contiguous to those encoding for 5,6-LAM, kamB and kamC, were identified to be responsible for the ATP-dependent reactivation of 5,6-LAM. The two gene products co-purified after being co-expressed in E. coli and were determined to form a complex, designated as KamBC. Aerobic UV-visible experiments showed that KamBC with ATP/Mg2+ was able to attenuate the rate of hydroxocobalamin formation and hence 5,6-LAM inactivation. Under anaerobic conditions, the presence of KamBC and ATP/Mg2+ prevented 5,6-LAM inactivation as time-resolved UV-visible spectra showed accumulation of the cob(II)alamin species and no detectable levels of hydroxocobalamin. KamBC displayed a basal level of ATPase activity that increased by 35% when coupled to 5,6-LAM turnover with b-L-lysine. We showed through steady-state kinetic assays that KamBC restored activity of 5,6-LAM by exchanging the damaged cofactor for free AdoCbl, and that activity was dependent on ATP hydrolysis. The functionality of KamBC was tested on 4,5-OAM, a homolog of 5,6-LAM, and it was determined that KamBC is specific for 5,6-LAM as there was no change in hydroxocobalamin formation when the reaction mixture of 4,5-OAM and D-ornithine also contained KamBC and ATP/Mg2+. The amino acid sequence of KamBC suggests that it is structurally distinct from other AdoCbl-dependent enzyme reactivating factors. Thus, this new metallochaperone system adds to the list of proteins that have evolved to maintain the cellular activity of AdoCbl-dependent enzymes. 136 5.2 Experimental Procedures 5.2.1 Materials F. nucleatum subsp. nucleatum (ATCC 25586) genomic DNA was from the American Tissue Culture Collection (Manassas, VA, USA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). Q5â High-Fidelity DNA polymerase and restriction enzymes were from New England Biolabs (Ipswich, MA, USA). XL-1 Blue competent cells were from Agilent Technologies (Santa Clara, CA, USA). Rosetta(DE3)pLysS competent cells were from EMD Biosciences (Burlington, MA, USA). The E.Z.N.A Plasmid DNA Mini Kit was from VWR (Radnor, PA, USA). The HisTrap-FF column and VivaspinÒ 20, 30 MWCO centricons were from Cytiva (Marlborough, MA, USA). Bovine alpha-Thrombin was from Prolytix (Essex Junction, VT, USA). The Econo-Pac 10 DG size exclusion column and Mini-PROTEAN TGX gels (4-15%) were from Bio-Rad (Hercules, CA, USA). The PageRuler pre-stained protein ladder, 12-14 kDa MWCO dialysis membrane tubing, PierceTM BCA protein assay, malachite green oxalate, ammonium molybdate(para)tetrahydrate, and Triton X-100 were from Thermo Fisher Scientific (Waltham, MA, USA). The HiLoad 16/60 Superdex 200 pg (1.6 x 60 cm) column was from GE Healthcare Technologies (Chicago, IL, USA). PLP, AdoCbl, ADP, ATP, AMPPNP, D-ornithine, NADP+, and High Molecular Weight (12-200 kDa) Markers Kit for gel filtration were from Sigma (St. Louis, MO, USA). Dithiothreitol was from TCI Chemicals (Portland, OR, USA). b-L-lysine ((3S)-3,6-diaminohexanoic acid) was purchased from CHEMspace (Monmouth Junction, NJ, USA). All other chemicals were purchased from Thermo Fisher Scientific and were of the highest grade available. 5.2.2 Cloning and expression of 5,6-LAM, KamBC, and DAHDH Ornithine 4,5-aminomutase (4,5-OAM) was cloned and expressed following a previously published protocol.113 The genes encoding for lysine 5,6-aminomutase (5,6-LAM; Fn1863/Fn1862), KamBC (Fn1865/Fn1864), and erythro-3,5-diaminohexanoate dehydrogenase (DAHDH; Fn1867) were amplified from the genomic DNA of F. nucleatum strain ATCC 25586 with Q5â High-Fidelity DNA polymerase using the primers listed in Table 5.1. 137 Table 5.1 Forward and reverse primers used in PCR to amplify 5,6-LAM, KamBC, and DAHDH.Name Sequence 5,6-LAM Forward 5′-G GAA TTC CAT ATG GGA AAA TTG GAT CTT GAT TGG-3′ 5,6-LAM Reverse 5′-CG GGA TCC TTA TTT ATT CAT ACC TCT TTG AAC C-3′ KamBC Forward 5′-G GAA TCC CAT ATG TTA GAT ACA TAT AAG TTT ATT GAG AAA TAT AAA AG-3′ KamBC Reverse 5′-CG GGA TCC TTA TTG TTC CTC CCA TTC-3′ DAHDH Forward 5′-G GAA TCC CAT ATG AAA AAA GGT TGT AAA TAC GGA AC-3′ DAHDH Reverse 5′-CG GGA TCC TTA TAA ATA TAT TTC ATT AAA TAT TTC TCT TAA CAC TGC-3′ The NdeI and BamHI restriction sites are shown in bold. The PCR mixture contained Q5 DNA polymerase (0.02 U/µL), Q5 reaction buffer, dNTPs (200 µM), F. nucleatum genomic DNA (1.0 ng/mL), forward primer (0.5 µM), and reverse primer (0.5 µM) for a total reaction volume of 50 µL. The PCR cycling parameters for each gene were as described in Table 5.2. Table 5.2 PCR cycling parameters for amplification of 5,6-LAM, KamBC, and DAHDH. 5,6-LAM KamBC DAHDH Cycles Temperature (°C) Time (s) Temperature (°C) Time (s) Temperature (°C) Time (s) 1 98 30 98 30 98 30 30 98 10 98 10 98 10 69 30 68 30 66 30 72 75 72 30 72 75 1 72 120 72 120 72 120 138 Each PCR product was cleaved with NdeI and BamHI and ligated into the pET-15b vector, which was also digested with the same restriction enzymes. The ligation mixture was used to transform E. coli strain XL-1 Blue competent cells and the cell mixture was plated on LB-agar plates supplemented with ampicillin (100 µg/mL). The plasmid from a transformed cell was isolated using an E.Z.N.A Plasmid DNA Mini Kit. DNA sequencing by the Sequencing and Bioinformatics Consortium at UBC Vancouver confirmed the presence of the correct coding sequence in the pET-15b plasmid. Each plasmid construct was used to transform E. coli strain Rosetta(DE3)pLysS for protein expression. A starter culture was prepared by inoculating a single transformed colony into 100 mL of LB media supplemented with ampicillin (100 µg/mL) and chloramphenicol (34 µg/mL). The starter culture was incubated for approximately 16 h at 37 °C. Aliquots (10 mL) of the starter culture were added to 1 L of TB supplemented with ampicillin (100 µg/mL) and chloramphenicol (34 µg/mL). The culture was incubated at 37 °C at 200 rpm until the OD600 was ~0.6. IPTG was added to cell cultures at a final concentration of 0.2 mM. The cultures were incubated for approximately 16 h at 25 °C at 200 rpm. Cell cultures were harvested by centrifugation at 4355 g for 15 min at 4 °C and cell pellets were pooled together and stored at -80 °C until purification. 5.2.3 Enzyme purification of 5,6-LAM, KamBC, and DAHDH 4,5-OAM was purified by a previously published protocol.113 5,6-LAM, KamBC, and DAHDH were purified using the same general protocol, except that the DAHDH purification contained 0.15 M NaCl in the lysis and elution buffers. All purification steps were carried out at 4 °C. Cells overexpressing the respective protein (15-30 g wet weight) were resuspended in 150 mL of 50 mM Tris-HCl pH 7.5 containing 5 mM imidazole and protease inhibitors benzamidine (0.5 mM) and phenylmethylsulfonyl fluoride (10 µM). The cell suspension was sonicated on ice at 30% power for 8 s at 1 min intervals for 60 min, followed by centrifugation at 39 191 g for 60 min at 4 °C. The supernatant was pooled and then applied to a 5 mL HisTrap-FF column equilibrated with 50 mM Tris-HCl pH 7.5 and 5 mM imidazole. Protein was eluted from the column using a step gradient: 5 column volumes (CV) of 96% Buffer A (50 mM Tris-HCl pH 7.5) and 4% Buffer B (50 mM Tris-HCl pH 7.5 and 0.5 M imidazole), 5 CV of 92% Buffer A and 8% Buffer B, 5 CV of 40% Buffer A and 60% Buffer B, and 5 CV of 100% Buffer B. Elution of the protein was monitored at 280 nm. Fractions containing protein were pooled and concentrated to ~10 mL using 139 a 30 kDa MWCO centricon. The proteins were each dialyzed against 50 mM Tris-HCl pH 7.5, 0.1 M NaCl, 1 mM EDTA, and 2 mM b-mercaptoethanol for 16 h at 4 °C using 12-14 kDa MWCO dialysis tubing. PLP (10 µM) was added to the dialysis buffer for the dialysis of 5,6-LAM. Thrombin was added to DAHDH in a 400:1 protein:thrombin (mg:mg) ratio prior to dialysis to cleave the N-terminal His6-tag. The dialysate of DAHDH was re-applied to the HisTrap-FF column equilibrated with 50 mM Tris-HCl pH 7.5 and 10 mM imidazole to separate the thrombin-cleaved protein from the His6-tagged protein. The column was washed with 3 column volumes of equilibration buffer and the flow through was collected. SDS-PAGE was performed to assess the purity of each protein. Stocks of the proteins were prepared in 20% glycerol and stored at -80 °C. The concentrations of 5,6-LAM, DAHDH, and KamBC were determined by measuring the absorbance at 280 nm using an extinction coefficient calculated from the PierceTM BCA protein assay using bovine serum albumin as a standard (e = 54 947 M-1 cm-1 for 5,6-LAM, 53 397 M-1 cm-1 for KamBC, and 10 558 M-1 cm-1 for DAHDH). 5.2.4 Gel filtration chromatography Size-exclusion chromatography with 5,6-LAM, KamBC, and DAHDH was performed using a HiLoad 16/60 Superdex 200 pg (1.6 x 60 cm) column equilibrated with 50 mM potassium phosphate pH 7.2 and 0.15 M NaCl buffer. Aliquots (500 µL) of the protein standards ovalbumin, aldolase, ferritin, thyroglobulin, and blue dextran were loaded onto the column at concentrations of 4 mg/mL, 4 mg/mL, 0.3 mg/mL, 5 mg/mL, and 1 mg/mL, respectively. The protein standards were eluted at a flow rate of 1 mL/min using an isocratic gradient of the equilibration buffer. The elution volume of each protein (Ve) was measured and the proportion of pores available to the molecule (Kav) was determined using Equation 5.1. Equation 5.1 V7 = V& + 𝐾#8(V9 − V&) where Ve is the elution volume, Vo is the void volume (elution volume of blue dextran), Kav is the proportion of the pores available to the molecule, and Vt is the total column volume. 140 The calculated Kav values of the standard proteins were plotted against the logarithm of the molecular weight (MW) or Stokes radius (Rs) to produce a calibration curve.192 Aliquots (500 µL) of 5,6-LAM, KamBC, or DAHDH at ~5 mg/mL were loaded onto the column and eluted using an isocratic gradient to determine the elution volumes. Equation 5.1 was used to determine Kav, and the molecular weight or hydrodynamic radius was determined from the calibration curve for each protein sample. 5.2.5 Aerobic UV-visible spectroscopy Aerobic spectral assays were carried out on a PerkinElmer Lambda 25 UV-visible spectrophotometer. Each reaction (1 mL) contained 10 µM 5,6-LAM, 10 µM PLP, 10 µM AdoCbl, and 5 mM dithiothreitol (DTT) in 100 mM NH4EPPS pH 8.5 buffer. Absorbance spectra were recorded with and without the addition of 10 µM KamBC, 5 mM MgCl2, and 5 mM ADP or ATP. A reference spectrum was recorded with every component in the reaction mixture except for b-L-lysine. The reactions were initiated with 2.5 mM b-L-lysine and scans were collected from 300-600 nm every 1 min over 15 min at 25 °C. Assays containing 4,5-OAM were performed under the same conditions except 4,5-OAM was substituted for 5,6-LAM and D-ornithine was substituted for b-L-lysine. 5.2.6 Anaerobic UV-visible spectroscopy Anaerobic spectral assays were performed on a PerkinElmer Lambda 265 UV-visible spectrophotometer housed in a Belle Technology glove box under a 99.998% N2 atmosphere. Buffer (100 mM NH4EPPS pH 8.5) was degassed with nitrogen for 2 h and then brought into the glove box where it was allowed to equilibrate for 18 h in an oxygen-free environment. Solid PLP, AdoCbl, DTT, b-L-lysine, ADP, ATP, AMPPNP, and MgCl2 were brought into the glove box and dissolved in anaerobic buffer. Concentrated samples (2 mL) of 5,6-LAM and KamBC were introduced into the glove box and gel-filtered over a 10 mL size-exclusion column equilibrated with anaerobic buffer. Each reaction (1 mL) contained 10 µM 5,6-LAM, 10 µM PLP, 10 µM AdoCbl, and 5 mM DTT in 100 mM NH4EPPS pH 8.5 buffer. The UV-visible spectra were recorded with and without 10 µM KamBC, 5 mM MgCl2, and 5 mM ADP, ATP, or AMPPNP. A 141 reference spectrum was recorded with all components of the reaction mixture except for b-L-lysine. The reactions were initiated with 2.5 mM b-L-lysine and spectra were collected from 300-600 nm every 1 min over 15 min at 25 °C. The rate of inactivation of the AdoCbl cofactor of 5,6-LAM was determined by fitting a single exponential growth function (Equation 5.2) to a plot of the absorbance change at 355 nm versus time using a non-linear least squares regression analysis in the computer program Origin (version 8.5). The rate of inactivation (kinactivation) was determined from the inverse of t1. Equation 5.2 y = A11e$/947 + y2 where y is the absorbance at 355 nm, A1 is the amplitude, x is the time, t1 is the inverse of the rate of inactivation, and y0 is the final absorbance. 5.2.7 Steady-state kinetic assays measuring 5,6-LAM turnover 5,6-LAM activity was measured using a coupled spectrophotometric assay with DAHDH. DAHDH catalyzes the NADP-dependent oxidative deamination of erythro-3,5-diaminohexanoate, the product of 5,6-LAM turnover. The kinetic assays (1 mL) were performed in triplicate in 100 mM NH4EPPS pH 8.5 at 25 °C. The reaction mixture contained 100 nM 5,6-LAM, 10 μM PLP, 10 μM AdoCbl, 5 μM DAHDH, 0.15 mM NADP+, and varying amounts of b-L-lysine (5-250 μM). A reference spectrum of the reaction mixture containing all components of the reaction except for b-L-lysine was recorded for every reaction, prior to the addition of b-L-lysine. The initial velocity of the reaction was determined by the absorbance change at 340 nm over 60 s using an extinction coefficient of 6.22 mM-1 cm-1. Kinetic parameters were determined by fitting the Michaelis-Menten equation (Equation 2.1) to the data using a non-linear least squares regression analysis using the computer program Origin (version 8.5). The same coupled assay with DAHDH was used to determine if KamBC could reactivate 5,6-LAM containing the hydroxocobalamin cofactor. For this experiment, a 2 mL reaction containing 50 μM 5,6-LAM, 50 μM PLP, 50 μM AdoCbl, and 2.5 mM b-L-lysine in 100 mM NH4EPPS pH 142 8.5 was incubated in the dark at 25 °C for 2 h to generate inactive 5,6-LAM. The reaction mixture was dialyzed for approximately 16 h against 50 mM Tris-HCl pH 7.5 to remove erythro-3,5-diaminohexanoate. The concentration of inactivated 5,6-LAM was determined from the absorbance peak at 355 nm using an extinction coefficient of 27.5 mM-1 cm-1.193 Inactive 5,6-LAM (2 μM) was then added to a 1 mL reaction containing 10 μM DAHDH, 0.15 mM NADP+, and 1 mM b-L-lysine in 100 mM NH4EPPS pH 8.5 buffer. A reference spectrum was recorded for the reaction mixture containing everything except b-L-lysine. The reaction was then initiated with the addition of 1 mM b-L-lysine. The initial velocity of the reaction was determined by monitoring the change in absorbance at 340 nm over 5 min as described above. The reaction was repeated with the addition of 10 μM AdoCbl, 10 μM KamBC, 5 mM MgCl2, and 5 mM ADP, ATP, or AMPPNP. All reactions were completed in triplicate with the average absorbance traces plotted. 5.2.8 Steady-state kinetic assays measuring ATPase activity of KamBC ATPase activity of KamBC was determined using a procedure modified from the malachite green assay to monitor the release of inorganic phosphate (Pi).194,195 The reactions were performed in triplicate at 37 °C in a total volume of 50 µL in 100 mM Tris-HCl pH 8.5 containing 5 mM ATP and 5 mM MgCl2 with and without 1 mM b-L-lysine or D,L-lysine, 1 µM 5,6-LAM, and 10 µM AdoCbl. The reactions were initiated with 1 µM of KamBC. Each reaction was quenched at different time points (10, 20, 40, and 60 min) using 800 µL of a colour reagent (0.034% malachite green-HCl, 1.05% ammonium molybdate, 0.1% Triton X-100, and 1N HCl). After 1 min, 100 µL of 34% sodium citrate was added to each reaction tube, and then the reaction mixture was incubated for 40 min. Blank samples were prepared with all the reaction components except for the addition of enzyme(s) to correct for background absorbance due to non-enzymatic ATP hydrolysis. Absorbance values were measured at 650 nm using a PerkinElmer Lambda 25 UV-visible spectrophotometer, and the initial velocity of the reaction was determined from a standard curve of KH2PO4 (0-200 µM). The rate of ATP hydrolysis by KamBC was determined from a linear fit of the data using a linear regression analysis in the computer program Origin (version 8.5). 143 5.2.9 Native-PAGE Native-PAGE was performed to determine if KamBC forms a complex with the inactive form of 5,6-LAM. Inactivated 5,6-LAM was prepared by reacting 50 µM 5,6-LAM, 50 µM PLP, and 50 µM AdoCbl with 2.5 mM b-L-lysine in 50 mM NH4EPPS pH 8.5 in the dark for 2 h. The reaction mixture was then dialyzed for 16 h against 50 mM Tris-HCl pH 7.5 to remove excess AdoCbl. In a total volume of 20 µL, 20 µM of KamBC was added to 20 µM of inactivated 5,6-LAM, and the mixture was incubated at 37 °C. After 10 min, 20 mM ADP and 20 mM MgCl2 were added to the mixture which was then further incubated at 37 °C for 1.5 h. Samples containing only inactivated 5,6-LAM or KamBC with ADP/Mg2+ were subjected to the same conditions. PAGE (4-15% gel) was performed under non-denaturing conditions in the presence of 1 mM ADP and 1 mM MgCl2. Gel electrophoresis was performed at 100 V for 6 h at 4 °C. 5.3 Results 5.3.1 Properties of purified 5,6-LAM, KamBC, and DAHDH The genes for 5,6-LAM, KamBC, and DAHDH were cloned into a pET-15b expression vector which added a sequence encoding a N-terminal His6-tag with a thrombin cleavage site to the open reading frame. The recombinant proteins were expressed in E. coli and purified through Ni-NTA affinity chromatography. The apparent molecular weight of each protein was determined using SDS-PAGE as shown in Figure 5.1. Expectedly, the two subunits for 5,6-LAM, KamD and KamE, copurified with apparent molecular weights of ~57 kDa (KamD) and 29 kDa (KamE). These molecular weights correspond to the calculated molecular weights for the KamD and KamE subunits of 59 413 Da and 28 990 Da, respectively, based on their amino acid sequences. SDS-PAGE analysis for KamBC showed that KamB and KamC copurify and form a complex. The molecular weights determined from the gel were determined to be 38 kDa and 56 kDa for KamB and KamC, respectively, which are similar to the calculated molecular weights of 37 795 Da for KamB and 56 485 Da for KamC. DAHDH appeared as one band on the SDS-PAGE gel with an apparent mass of 37 kDa, which is comparable to the calculated molecular weight of the protein without the His6-tag (35 614 Da). 144 Figure 5.1 SDS-PAGE gel of 5,6-LAM, KamBC, and DAHDH. (A) SDS 12% polyacrylamide gel showing purification of 5,6-LAM by Ni-NTA affinity chromatography. Lane 1, PageRuler pre-stained protein ladder; Lane 2, crude extract from 5,6-LAM overexpressing Rosetta(DE3)pLysS cells; Lane 3, purified 5,6-LAM eluted from the Ni-NTA column. (B) SDS 10% polyacrylamide gel showing purification of KamBC by Ni-NTA affinity chromatography. Lane 1, PageRuler pre-stained protein ladder; Lane 2, crude extract from KamBC overexpressing Rosetta(DE3)pLysS cells; Lane 3, flow-through from the Ni-NTA column; Lane 4, purified KamBC eluted from the Ni-NTA column. (C) SDS 12% polyacrylamide gel showing purification of DAHDH by Ni-NTA affinity chromatography. Lane 1, PageRuler pre-stained protein ladder; Lane 2, crude extract from DAHDH overexpressing Rosetta(DE3)pLysS cells; Lane 3, flow-through from the Ni-NTA column; Lane 4, purified DAHDH eluted from the Ni-NTA column after thrombin cleavage. 5.3.2 Gel filtration chromatography Each of the proteins (5,6-LAM, KamBC, and DAHDH) was subjected to size-exclusion chromatography to determine their quaternary structure. A standard curve was generated using the following proteins: ovalbumin (44 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa) (Figure 5.2). Based on the elution volume, the estimated molecular weight of 5,6-LAM was 217 kDa. With a monomeric mass of 88.4 kDa, the estimated mass is consistent with an a2b2 heterodimer assembly of 5,6-LAM.114 KamBC eluted from the column as one peak to give a total mass of 128 kDa, which is slightly higher than an ab complex with a calculated mass of 94.3 kDa. 180 kDa130 kDa100 kDa70 kDa55 kDa40 kDa35 kDa25 kDa15 kDaLane 1 Lane 2 Lane 3180 kDa130 kDa100 kDa70 kDa55 kDa40 kDa35 kDa25 kDa15 kDa10 kDaLane 1 Lane 2 Lane 3 Lane 4180 kDa130 kDa100 kDa70 kDa55 kDa40 kDa35 kDa25 kDa15 kDa10 kDaLane 1 Lane 2 Lane 3 Lane 4A B C 145 DAHDH eluted as a homotetramer based on the monomeric mass of 35.6 kDa and a total molecular weight of 175 kDa. The quaternary structures of each of the proteins are summarized in Table 5.3. Table 5.3 Quaternary structure determination of 5,6-LAM, KamBC, and DAHDH based on estimated molecular weights by size-exclusion chromatography.Enzyme Total Mr MW of Subunit Quaternary Structure 5,6-LAM 217 88.4 a2b2 heterodimer KamBC 128 94.3 ab monomer DAHDH 175 35.6 a4 homotetramer Figure 5.2 Estimated molecular weights of 5,6-LAM, KamBC, and DAHDH from gel filtration chromatography. A plot of the Kav versus the logarithm of molecular weight of different proteins based on their elution volume. The standard proteins are shown in grey, 5,6-LAM is shown as a black closed circle, KamBC is shown as a black open circle, and DAHDH is shown as a black square. The standard proteins for gel filtration chromatography are a globular shape, so the determined molecular weights based on elution volume may be a good estimate if the protein of interest is a similar shape. However, if the protein of interest varies from a spherical protein, it can result in an 146 overestimation of the molecular weight. Because of this, the Stokes radius (Rs) can be a better estimate than molecular weight based on the elution profile from gel filtration.196 From a calibration plot of the standard proteins, the calculated Rs values of 5,6-LAM, KamBC, and DAHDH were determined to be 5.2, 4.3, and 4.8 nm, respectively, as shown in Figure 5.3. In comparison to Figure 5.2, both KamBC and DAHDH appear to be smaller in size with respect to the standard proteins, which suggests that they may form a more extended conformation in comparison to the globular shape of the standard proteins. Figure 5.3 Estimated hydrodynamic radius of 5,6-LAM, KamBC, and DAHDH. A plot of the logarithm of the Stokes radius versus the Kav of different proteins. The standard proteins are shown in grey, 5,6-LAM is shown as a black closed circle, KamBC is shown as a black open circle, and DAHDH is shown as a black square. 5.3.3 UV-visible spectroscopy After purification, 5,6-LAM had a slight yellow colour, indicating the presence of the PLP cofactor. A UV-visible absorbance spectrum of 5,6-LAM in Figure 5.4 (A) revealed an absorbance maximum at 426 nm with a small shoulder at 330 nm, confirming the presence of the PLP cofactor in primarily the ketoenamine tautomeric form. The concentration of PLP-bound 5,6-LAM, determined from the peak at 426 nm, was calculated to be 34 µM using an extinction coefficient of 6.6 mM-1 cm-1. In comparison, the concentration of apo-5,6-LAM determined from the PierceTM 147 protein assay was 133 µM. Thus, the ratio of PLP-bound 5,6-LAM to apo-5,6-LAM was 1:4. This result indicates that only 25% of 5,6-LAM contained the PLP cofactor. When 5,6-LAM was complexed with both PLP and AdoCbl (holo-5,6-LAM), the protein was a slight red colour and the absorbance spectrum exhibited multiple absorbance peaks that correspond to both the PLP (426 nm) and the AdoCbl (337, 378, and 525 nm) cofactors as shown in Figure 5.4 (B). Figure 5.4 PLP and AdoCbl absorbance spectra of 5,6-LAM. (A) UV-visible absorbance spectrum of 5,6-LAM with the PLP absorbance maximum at 426 nm. (B) Spectrum of 5,6-LAM with PLP and AdoCbl with peaks at 337, 378, 426, and 525 nm. Changes in the absorbance spectra of holo-5,6-LAM were followed upon the addition of b-L-lysine. To observe the formation of intermediates more clearly, these absorbance changes were recorded as difference spectra. As shown in panel A of Figure 5.5, the first spectrum recorded at 1 min following the addition of substrate shows the rapid inactivation of 5,6-LAM in the presence of O2. Subsequent absorbance spectra collected over the next 15 min were identical, indicating that the enzyme completely converted into the inactive form. Inactivation in the presence of O2 occurs quickly because oxidation of the cob(II)alamin intermediate prevents the regeneration of AdoCbl for subsequent rounds of catalysis. The appearance of a major peak at 355 nm as well as two minor peaks at 500 nm and 536 nm are characteristic of the formation of hydroxocobalamin.121 When KamBC alone or in combination with ADP/Mg2+ were added to the reaction mixture, there was still an accumulation of the inactive cofactor as indicated by the increase at 355 nm with little absorbance changes over time (Figure 5.5 (B, C)). In the reaction with KamBC and ATP/Mg2+, A B 148 the formation of hydroxocobalamin occurred more slowly over 15 min, indicating that in aerobic conditions, KamBC in the presence of ATP/Mg2+ slows the rate of substrate-induced inactivation of 5,6-LAM (Figure 5.5 (D)). Figure 5.5 Aerobic absorbance spectra of 5,6-LAM with b-L-lysine in the absence and presence of KamBC alone and with ADP or ATP. Aerobic UV-visible absorbance spectra of (A) a holo-5,6-LAM solution containing 10 µM 5,6-LAM, 10 µM PLP, 10 µM AdoCbl, and 5 mM DTT in 100 mM NH4EPPS pH 8.5 buffer. Absorbance spectra were also recorded with the addition of (B) 10 µM KamBC, (C) 10 µM KamBC, 5 mM MgCl2, and 5 mM ADP, and (D) 10 µM KamBC, 5 mM MgCl2, and 5 mM ATP. All spectra were recorded every minute for 15 min after the addition of 2.5 mM b-L-lysine. The arrows at 355 nm indicate the direction of absorbance changes over time. Given that 5,6-LAM readily inactivates in the presence of O2, absorbance spectra were also recorded anaerobically. In Figure 5.6 (A), similar to the spectra recorded in aerobic conditions, A B C D 149 addition of b-L-lysine to 5,6-LAM caused the formation of hydroxocobalamin over time. This inactive form of 5,6-LAM was readily observed by absorbance increases at 355, 505, and 535 nm. These changes coincided with absorbance decreases at 310 nm and 470 nm, corresponding to the cob(II)alamin intermediate. Thus, following homolysis of the Co-C bond, the reaction of 5,6-LAM and b-L-lysine rapidly forms cob(II)alamin before undergoing suicide inactivation. The addition of KamBC and ADP/Mg2+ or AMPPNP/Mg2+ (a non-hydrolyzable analog of ATP) to the reaction mixture resulted in similar spectral changes to the reaction with 5,6-LAM alone (Figure 5.6 (B, C)). However, when the reaction of 5,6-LAM and b-L-lysine contained KamBC and ATP/Mg2+, there was no formation of hydroxocobalamin as there was no increase in absorbance at 355, 505, or 535 nm. Instead, the formation of cob(II)alamin was more readily observed as seen with absorbance increases at 310 nm and 470 nm over time (Figure 5.6 (D)). There was also a decrease in absorbance at 528 nm due to homolysis of the Co-C bond during catalysis. These data show that the presence of KamBC and ATP/Mg2+ helps stabilize the formation of the cob(II)alamin intermediate of 5,6-LAM. Because the formation of hydroxocobalamin was slower under anaerobic conditions compared to aerobic conditions, the inactivation rates of 5,6-LAM were determined by plotting the absorbance change at 355 nm versus time. The reaction of 5,6-LAM with b-L-lysine produced a kinactivation value of 0.34 ± 0.01 min-1 (Table 5.4). For the conditions with KamBC, MgCl2, and AMPPNP or ADP, the kinactivation values were comparable at 0.24 ± 0.05 min-1, and 0.32 ± 0.03 min-1, respectively. Table 5.4 Anaerobic rates of inactivation of 5,6-LAM with b-L-lysine in different reaction conditions determined from the change in absorbance at 355 nm over time.Reaction Conditions kinactivation (min-1) 5,6-LAM + b-L-lysine 0.34 ± 0.01 5,6-LAM + KamBC + MgCl2 + AMPPNP + b-L-lysine 0.24 ± 0.05 5,6-LAM + KamBC + MgCl2 + ADP + b-L-lysine 0.32 ± 0.03 150 Figure 5.6 Anaerobic absorbance spectra of 5,6-LAM with b-L-lysine in the absence and presence of KamBC with AMPPNP, ADP, or ATP. Anaerobic UV-visible absorbance spectra of (A) a holo-5,6-LAM solution containing 10 µM 5,6-LAM, 10 µM PLP, 10 µM AdoCbl, and 5 mM DTT in 100 mM NH4EPPS pH 8.5 buffer. Absorbance spectra were also recorded with the addition of 10 µM KamBC, 5 mM MgCl2, and 5 mM (B) AMPPNP, (C) ADP, or (D) ATP. All spectra were recorded every minute for 15 min after the addition of 2.5 mM b-L-lysine. The arrows at 310, 355, 470, 505, 528, and 535 nm indicate the direction of absorbance changes over time. Panels A, B, and C contain insets of the rate of hydroxocobalamin formation measured by the absorbance increase at 355 nm over time. Panel D contains insets of the rate of cob(II)alamin formation at 310 nm and the rate of homolysis at 528 nm over time. 4,5-OAM and 5,6-LAM are the only two members of the aminomutase family of AdoCbl-dependent enzymes. Given that KamBC was able to slow the inactivation of 5,6-LAM in the presence of ATP/Mg2+, the question arose of whether it could do the same with its homolog, 4,5-OAM. To test this, the same spectral studies were implemented using 4,5-OAM and its substrate, C A B D 151 D-ornithine. Figure 5.7 (A) shows the absorbance spectra recorded over time after the addition of D-ornithine to 4,5-OAM under aerobic conditions. The conversion of AdoCbl to hydroxocobalamin in 4,5-OAM was apparent by increases in absorbance at 357, 504, and 539 nm. When the reaction mixture also included KamBC and ATP/Mg2+, there was no discernable difference in the time-resolved absorbance spectra (Figure 5.7 (B)). These results suggest that KamBC is specific for 5,6-LAM, and perhaps 4,5-OAM has its own reactivation system. Figure 5.7 Aerobic absorbance spectra of 4,5-OAM with D-ornithine in the absence and presence of KamBC and ATP. Aerobic UV-visible absorbance spectra of (A) 10 µM 4,5-OAM, 10 µM PLP, 10 µM AdoCbl, and 5 mM DTT in 100 mM NH4EPPS pH 8.5 buffer following the addition of 2.5 mM D-ornithine. (B) As for panel A, but with the addition of 10 µM KamBC, 5 mM MgCl2, and 5 mM ATP to the 1 mL reaction. Absorbance spectra were collected every minute for 15 min following the addition of 2.5 mM D-ornithine. The arrows at 357, 504, and 539 nm indicate the direction of absorbance changes over time. 5.3.4 Steady-state kinetic analysis of 5,6-LAM turnover A coupled spectrophotometric assay with 5,6-LAM and DAHDH was used in order to determine the macroscopic kinetic constants associated with 5,6-LAM turnover with b-L-lysine. Given that DAHDH catalyzes the NADP-dependent oxidative deamination of erythro-3,5-diaminohexanoate, an absorbance increase at 340 nm following the conversion of NADP+ to NADPH can be used to determine the initial rate of 5,6-LAM turnover. A Michaelis-Menten plot was generated from the initial velocity of the reaction versus increasing concentrations of b-L-lysine (Figure 5.8). The B A 152 kinetic parameters kcat and KM were calculated to be 0.19 ± 0.01 s-1 and 13.8 ± 0.9 µM, respectively. The catalytic efficiency (kcat/KM) was 13900 ± 1160 s-1 M-1 (Table 5.5). Table 5.5 Steady-state kinetic parameters associated with the production of erythro-3,5-diaminohexanoate from the reaction of 5,6-LAM with b-L-lysine.kcat (s-1) KM (µM) kcat/KM (s-1 M-1) 0.19 ± 0.01 13.8 ± 0.9 13900 ± 1160 Figure 5.8 Determination of steady-state kinetic parameters associated with erythro-3,5-diaminohexanoate production for 5,6-LAM. A plot of the initial velocity versus the concentration of b-L-lysine was generated for the reaction of 5,6-LAM with b-L-lysine. The initial velocity was determined based on NADPH formation through a coupled assay with DAHDH. The coupled assay was then performed with inactive 5,6-LAM in the presence of AdoCbl and the absorbance change was monitored at 340 nm over 5 min. Incubation of inactive 5,6-LAM and AdoCbl with and without KamBC and AMPPNP/Mg2+ or ADP/Mg2+ resulted in no significant absorbance change, thus no detectable activity. As illustrated in Figure 5.9, reactivation of 5,6-LAM by KamBC was evident in the presence of free AdoCbl and ATP/Mg2+ due to an increase in absorbance at 340 nm over the time course of the assay with an initial rate of 29 ± 0.1 min-1. When 153 no additional AdoCbl was present in the reaction mixture, there was no activity under any of the conditions described above, indicating that free AdoCbl is required for reactivation of 5,6-LAM in the presence of KamBC and ATP/Mg2+. Figure 5.9 KamBC and ATP-dependent reactivation of inactive 5,6-LAM. Absorbance traces tracking NADPH formation at 340 nm from the coupled assay of 5,6-LAM with DAHDH. Each reaction contained inactivated 5,6-LAM, AdoCbl, DAHDH, NADP+, and β-L-lysine (black line). The reactions were repeated with the addition of KamBC, MgCl2, and AMPPNP (black dashed line), ADP (black dotted line), or ATP (grey line). 5.3.5 Steady-state kinetic analysis of KamBC ATPase activity The rate of ATP hydrolysis by KamBC was determined through the malachite green assay under different turnover conditions (Table 5.6). When KamBC was absent, no ATP hydrolysis activity was detected (Figure 5.10). When KamBC, ATP/Mg2+ and either 5,6-LAM or AdoCbl were in the reaction mixture with β-L-lysine, the ATPase activity was determined to be 2.4 ± 0.1 min-1 and 2.7 ± 0.1 min-1, respectively. These data indicate that KamBC exhibits a basal level of ATPase activity, regardless of if 5,6-LAM or AdoCbl are present. The reactions containing KamBC, ATP/Mg2+, 5,6-LAM, AdoCbl, and β-L-lysine or D,L-lysine caused a 1.5-fold increase in activity with rate constants of 3.7 ± 0.1 min-1 with β-L-lysine and 3.4 ± 0.2 min-1 with D,L-lysine. These data suggest 154 that 5,6-LAM turnover accelerates ATP hydrolysis by KamBC. The activity of KamBC is also specific for ATP as there was no detectable activity when GTP was substituted for ATP. Table 5.6 Rate of ATP hydrolysis with D,L-lysine or β-L-lysine in the presence and absence of KamBC, 5,6-LAM, and AdoCbl.KamBC (1 µM) 5,6-LAM (1 µM) Substrate (1 mM) AdoCbl (10 µM) Rate of ATP hydrolysis (min-1) + + D,L-lysine + 3.4 ± 0.2 + + β-L-lysine + 3.7 ± 0.1 + + β-L-lysine - 2.4 ± 0.1 + - β-L-lysine + 2.7 ± 0.1 - + β-L-lysine + ND ND – not detected Figure 5.10 Rate of ATP hydrolysis for KamBC in varying conditions. A plot of the inorganic phosphate (Pi) concentration versus time was generated for the ATPase assay in varying conditions: using D,L-lysine or β-L-lysine as a substrate, and with and without 5,6-LAM, AdoCbl, and KamBC. The initial velocity of each reaction was determined from the slope of a linear fit to the data. 155 5.3.6 Native-PAGE analysis Native-PAGE was performed to determine if inactive 5,6-LAM forms a stable complex with KamBC. As shown in Figure 5.11, when inactivated 5,6-LAM was incubated with KamBC and ADP/Mg2+, no complex formation was detected as no new bands appeared on the gel compared to when KamBC and 5,6-LAM were loaded onto the gel in isolation. Perhaps 5,6-LAM and KamBC only form a weak complex and can easily dissociate when in solution. Figure 5.11 Native-PAGE gel of 5,6-LAM and KamBC. 4-15% gel run under non-denaturing conditions in the presence of ADP/Mg2+. Lane 1, inactive 5,6-LAM; Lane 2, KamBC with ADP/Mg2+; Lane 3, inactive 5,6-LAM with KamBC and ADP/Mg2+. 5.4 Discussion 5.4.1 5,6-LAM undergoes suicide inactivation during catalysis AdoCbl-dependent isomerases initiate catalysis through cleavage of the Co-C bond of the enzyme-bound complex, generating cob(II)alamin and the highly reactive 5′-deoxyadenosyl radical. At the end of catalysis, AdoCbl is normally regenerated through the re-formation of the Co-C bond. In the case where the reactive radical species extinguishes itself, it is impossible to regenerate AdoCbl for subsequent turnover.125 Consequently, the cofactor is irreversibly oxidized to form hydroxocobalamin. This causes inactivation of the enzyme because hydroxocobalamin is tightly bound to the enzyme and is unable to be exchanged with free AdoCbl. Rapid inactivation of 5,6-Lane 1 Lane 2 Lane 3 156 LAM has been reported to occur in the presence of either D-lysine or β-L-lysine, with the latter inducing inactivation at least 25 times faster.121 Under our experimental conditions, the inactivation rate for 5,6-LAM with β-L-lysine under anaerobic conditions was calculated to be 0.34 ± 0.01 min-1. By comparison, AdoCbl-dependent methylmalonyl-CoA mutase undergoes substrate-induced suicide inactivation at a rate that is 50-fold lower, with an inactivation rate of 0.0072 ± 0.0003 min-1.126 Steady-state kinetic parameters associated with 5,6-LAM turnover were determined through a coupled spectrophotometric assay with DAHDH. We observed the same initial velocity for NADPH formation under aerobic and anaerobic conditions using otherwise identical assay conditions. This observation is consistent with early reports by Baker et al. noting that the active form of 5,6-LAM was maintained if erythro-3,5-diaminohexanoate was rapidly removed by DAHDH.101 The kcat, KM, and kcat/KM for 5,6-LAM using β-L-lysine as a substrate were determined to be 0.19 ± 0.01 s-1, 13.8 ± 0.9 µM, and 13900 ± 1160 s-1 M-1, respectively. Notably, the KM for β-L-lysine was 20-fold lower than the value determined by Baker et al. (0.3 mM).101 In 2003, Tang et al. determined the KM for β-L-lysine to be 8.7 ± 0.7 mM with kcat and kcat/KM values of 255 ± 6 min-1 and 29.3 ± 2.5 mM-1 min-1, respectively.197 The variation in these results is likely due to the methods and conditions of the experiments performed across these different studies. 5.4.2 Reactivation of 5,6-LAM requires KamBC, ATP, and AdoCbl Our results show that in the presence of ATP/Mg2+, KamBC (i) stabilizes the cob(II)alamin species and (ii) reactivates 5,6-LAM from its inactive form through exchange of the damaged cofactor for free AdoCbl. AMPPNP was not effective in the reactivation of 5,6-LAM or the stabilization of the cob(II)alamin intermediate, indicating that ATP hydrolysis is required for both functions. In fact, the measured ATPase activity of KamBC was accelerated by 35% when combined with 5,6-LAM catalysis with β-L-lysine. In contrast, the basal level of ATPase activity of the reactivating factor of glycerol dehydratase (an AdoCbl-dependent eliminase) was unchanged in the presence of the dehydratase.198 157 We speculate that the mechanism by which KamBC exchanges hydroxocobalamin for AdoCbl in 5,6-LAM may be similar to the reactivases of AdoCbl-dependent eliminases such as diol dehydratase or glycerol dehydratase. The reactivating factor associated with diol dehydratase or glycerol dehydratase uses ATP/Mg2+ to mediate the exchange of the enzyme-bound adenine-lacking cobalamin (hydroxocobalamin) for an adenine-containing cobalamin (AdoCbl).125,199 Because hydroxocobalamin binds tightly and irreversibly to its coenzyme, the affinity for hydroxocobalamin must be lowered for it to be exchanged with AdoCbl. It was postulated that diol dehydratase forms a complex with its reactivating factor in order to complete this transition, and the free energy required for this process may be provided through coupling with ATP hydrolysis.125 In the eliminase enzyme systems, complex formation between the reactivating factor and the enzyme occurs when the ADP-bound reactivating factor induces a conformational change of the inactive enzyme, causing a release of the hydroxocobalamin cofactor. In the exchange of ADP with free ATP, the complex dissociates and the apo-enzyme can be reconstituted with AdoCbl to regenerate active holo-enzyme.200 Perhaps KamBC acts analogously to the reactivating factors of the eliminases, but it is still to be determined the specific mechanism and conditions in which 5,6-LAM and KamBC form a complex to achieve reactivation of the inactive enzyme. 5.4.3 Metallochaperone systems of AdoCbl-dependent enzymes are structurally distinct Although the metallochaperone system of KamBC shares a similar function to the reactivases of other AdoCbl-dependent enzymes, KamBC is structurally distinct from these chaperones. In the genome of F. nucleatum, KamB is annotated as a hypothetical protein and does not share known sequence homology with other proteins. KamC is annotated as MutS, a mismatch DNA repair protein. An AlphaFold model of KamC revealed that it may form an elongated shape, which could explain why the calculated Mr of KamBC from the gel filtration experiment (128 kDa) was larger than the weight of an ab monomer (95 kDa).201 The model of KamC was compared to the crystal structure of MutS from Thermus aquaticus to determine any structural similarities (Figure 5.12). MutS from Thermus aquaticus is a large dimeric protein (811 amino acids in length) that is organized into five domains, three of which are structurally similar to KamC (domains III, IV, and V).202 Domain III is entirely a-helical and is central to the MutS structure as it is directly connected to domains II, IV, and V. Domain IV consists of an elongated pair of antiparallel a-helices with a 158 b-sheet portion on the distal end that binds heteroduplex DNA. Notably, KamC lacks this b-sheet portion present in domain IV. Domain V is an ATPase domain that belongs to the ABC ATPase superfamily which also includes DNA repair/maintenance proteins such as UvrA and RAD50.203,204 Figure 5.12 Structural comparison of MutS with KamC. (A) MutS crystal structure from Thermus aquaticus (PDB entry 1EWQ at 2.20 Å resolution) showing domains I-V coloured from the N- to the C- terminus.202 (B) AlphaFold model of KamC coloured yellow, orange, and red to display potential structural similarity to domains III, IV, and V, respectively, in MutS.201 In contrast, the reactivating factor of diol dehydratase and glycerol dehydratase consists of an a2b2 heterodimer. The a-subunit has a core ATPase domain that generates the driving force of reactivase activity and shares structural similarity with the actin superfamily of proteins that includes the Hsp70 family of molecular chaperones (Figure 5.13 (A)).205,206 The b-subunit is composed of a single domain that has a similar structure to the b-subunit of the respective dehydratase, which enables a subunit switch that facilitates the release of the damaged AdoCbl cofactor (Figure 5.13 (B)).206,207 Other reactivating factors associated with AdoCbl-dependent A B IV III I II V (ATPase) DNA binding 159 eliminases, such as ethanolamine ammonia lyase and glutamate mutase, also have ATPase domains similar to the actin superfamily of proteins.208 Figure 5.13 Structure of diol dehydratase reactivase and diol dehydratase. Crystal structure of (A) diol dehydratase reactivase (yellow; PDB entry 2D0P at 3.00 Å resolution) and diol dehydratase (grey; PDB entry 1DIO at 2.20 Å resolution) from Klebsiella oxytoca.206,207 The ATPase domain is shown in red, the b-subunits are shown in violet, and cyanocobalamin, an inactive form of AdoCbl, is shown as orange sticks. The AdoCbl-dependent mutases involved in carbon skeleton rearrangement of CoA-derived substrates differ from the eliminases in that they utilize a guanine nucleotide-binding protein (G-protein) chaperone belonging to the G3E family of P-loop GTPases.209 MeaB, the reactivating factor of methylmalonyl-CoA mutase, is a homodimer that consists of N- and C-terminal a-helical extensions as well as a central G domain that houses the GTPase active site.210 A large conformational change in this domain occurs when MeaB binds GTP, switching from a GDP-bound form to a GTP-bound form. This results in complex formation with methylmalonyl-CoA mutase and allows MeaB to reassemble the active enzyme and protect it against inactivation.209,210 The difference in structure between these ATP- and GTP-dependent reactivating factors A B 160 demonstrates that they have evolved to maintain the cellular activity of these AdoCbl-dependent enzymes. 5.4.4 KamBC is specific for 5,6-LAM The only other known member of the aminomutase family of AdoCbl-dependent enzymes is ornithine 4,5-aminomutase (4,5-OAM), which shares a similar structure and mechanism as 5,6-LAM. The structure of 5,6-LAM consists of a triosephosphate isomerase (TIM) barrel domain (a-subunit) that binds the PLP cofactor, and a Rossmann domain (b-subunit) that binds AdoCbl (Figure 5.14 (A)).114 4,5-OAM exists as a a2b2 heterodimer and is encoded in the L-ornithine fermentation operon by oraE (large b-subunit) and oraS (small a-subunit).211 Unlike 5,6-LAM, the Rossmann and TIM barrel domains of 4,5-OAM are present within one b-subunit (Figure 5.14 (B)).118 The domains interact when the heterodimer undergoes a domain swap in which the Rossmann domain of one b-subunit interacts with the TIM barrel of the adjacent b-subunit, and vice versa, forming two identical active sites.118 Figure 5.14 Structural comparison of 5,6-LAM and 4,5-OAM. Crystal structures of (A) 5,6-LAM (PDB entry 1XRS at 2.80 Å resolution) and (B) 4,5-OAM (PDB entry 3KP1 at 2.01 Å resolution).114,118 The a-subunits are shown in grey and pink, the b-subunits are shown in yellow and orange, PLP is shown in black, and AdoCbl is shown in red. In comparison to 5,6-LAM, 4,5-OAM is less prone to undergo substrate-induced suicide inactivation under anaerobic conditions during the isomerization reaction of D-ornithine to 2,4-A B TIM Barrel Rossmann Domain TIM Barrel Rossmann Domain TIM Barrel TIM Barrel Rossmann Domain Rossmann Domain 161 diaminopentanoate. However, in the presence of O2, the AdoCbl cofactor of 4,5-OAM is oxidized to form hydroxocobalamin, thus inactivating the enzyme. It was postulated that KamBC could also reactivate 4,5-OAM in the presence of ATP/Mg2+, given that 4,5-OAM and 5,6-LAM are structural homologs. Absorbance spectra of 4,5-OAM with D-ornithine over 15 min displayed no difference compared to the spectra recorded with the addition of KamBC and ATP/Mg2+; there was still a clear formation of hydroxocobalamin and no sign of reactivation of the damaged cofactor, indicating the KamBC is specific for 5,6-LAM. 5.4.5 OraT as a potential reactivating factor for 4,5-OAM Within the L-ornithine fermentation operon in Clostridium sticklandii, six of the seven genes (ord, ortA, ortB, oraS, oraE, and orr) have been experimentally associated with a function in the catabolic pathway (Figure 5.15).212 The first step of this pathway involves the interconversion of L-ornithine to D-ornithine by ornithine racemase (orr). D-ornithine is converted to 2,4-diaminopentanoate in the reaction catalyzed by the gene product of oraS and oraE, ornithine 4,5-aminomutase (4,5-OAM). NAD-dependent oxidative deamination by 2,4-diaminopentanoate dehydrogenase (DADH, encoded by ord) leads to the formation of 2-amino-4-ketopentanoate. The final step in the pathway is catalyzed by the gene product of ortA and ortB, 2-amino-4-ketopentanoate thiolase (AKPT) and involves thiolytic cleavage of 2-amino-4-ketopentanoate with coenzyme A (CoA) to form D-alanine and acetyl-CoA. The remaining gene in the operon, oraT, is yet to be characterized. However, its gene product OraT is likely a reactivating factor for 4,5-OAM. OraT shares 37% sequence similarity to glmL, the gene that separates the genes encoding for the subunits of AdoCbl-dependent glutamate mutase (glmE and glmS) in Clostridium cochlearium.212,213 The amino acid sequences of OraT and GlmL reveal that they contain an ATP-binding motif similar to that of the reactivating factor of the AdoCbl-dependent eliminases, indicating that they may be involved in the ATP-dependent reactivation of 4,5-OAM and glutamate mutase, respectively.208 162 Figure 5.15 Ornithine fermentation operon and metabolic pathway of L-ornithine degradation. 5.5 Concluding Remarks Within the lysine fermentation pathway of F. nucleatum, 5,6-LAM is an AdoCbl-dependent enzyme that undergoes suicide inactivation in the presence of its substrate, β-L-lysine. The gene products contiguous to those of 5,6-LAM, KamB and KamC, were yet to be characterized. Herein, biochemical evidence is provided of KamBC forming a complex and functioning as a reactivating factor to repair inactivated 5,6-LAM through ATP hydrolysis. Anaerobic UV-visible absorbance studies showed that in the presence of KamBC and ATP/Mg2+, there was no accumulation of the inactive form of AdoCbl, hydroxocobalamin, and instead, there was a stabilization of the cob(II)alamin species. Techniques such as electron paramagnetic resonance (EPR) spectroscopy could be used in the future to study this radical species. It was also determined that free AdoCbl was absolutely required in the reactivation of 5,6-LAM, suggesting that KamBC may act similarly to the reactivating factors of the eliminases. It could not be concluded whether KamBC and 5,6-LAM interact because native-PAGE experiments showed no sign of complex formation in the presence of ADP. Further research into the structure of KamBC through X-ray crystallography may deduce if and how these proteins form a complex together to prevent inactivation of 5,6-ord oraTortA ortB oraS oraE orrCOO-H2NNH2COO-H2NNH2 NH2 NH2COO-L-ornithine D-ornithine 2,4-diaminopentanoateO NH2COO-2-amino-4-ketopentanoateSCoAO+NH2COO-acetyl-CoAD-alanineCoANAD+H2ONADHNH3ornithineracemaseorr4,5-OAMoraSEDADHordAKPTortAB 163 LAM. Isothermal titration calorimetry (ITC) could also be implemented to investigate the protein-protein and protein-ligand interactions between 5,6-LAM, KamBC, and substrates. This information will further our knowledge of the evolution of AdoCbl-dependent enzyme chaperone systems and their necessity for the protection of their highly reactive and metabolically expensive cofactor. 164 Chapter 6: Conclusion Over the last two decades, there has been a growing body of research focused on Fusobacterium nucleatum and its association with a range of different human pathologies. Given this linkage to systemic disease, it is important to determine metabolic aspects of F. nucleatum that enable it to adapt to different ecological niches and, under some conditions, trigger or exacerbate disease. A more detailed understanding of essential metabolic enzymes may also lead to new antimicrobials for the treatment of F. nucleatum-associated infections. The enzymes studied in this thesis are involved in distinct metabolic pathways within F. nucleatum. Chapters 2 and 3 discussed serine synthase, one of the four enzymes involved in H2S production in F. nucleatum. This PLP-dependent enzyme catalyzes the reversible reaction of L-cysteine and H2O to form L-serine and H2S. Given that F. nucleatum lacks the complete biosynthesis pathway for L-serine production, perhaps serine synthase is the only route for which F. nucleatum obtains L-serine for further use in the production of amino acids, proteins, and phospholipids. L-serine is also one of the few amino acids that F. nucleatum utilizes for energy via fermentation. In the serine synthase catalytic mechanism, an aspartic acid residue (D232) is required to act as a general base to deprotonate a water molecule for nucleophilic attack. A cluster of surrounding hydrophobic residues (W99, F125, F148, and F234) increase the basicity of the D232 carboxylate side chain for it to achieve this function. Due to the hydrophobic nature of the active site, D232 has no hydrogen bonding partners; however, F148 may engage in aromatic-anion interactions with D232 to properly position the side chain carboxylate for catalysis. While the hydrophobic active site residues also help to shield the PLP from the solvent, they restrict the number of nucleophiles that can react with the a-aminoacrylate intermediate. Single- and double- site substitutions of the phenylalanine residues (F125A, F148A, F234A, F125A/F234A, and F148A/F234A) allowed for the expansion of the substrate scope of serine synthase as thiol-based nucleophiles 2-mercaptoethanol and thiophenol reacted with the a-aminoacrylate intermediate to presumably form b-substituted amino acids. Therefore, these mutants allow for serine synthase to act as a biocatalytic tool for noncanonical amino acid synthesis. 165 Chapter 4 focused on lanthionine synthase, another PLP-dependent enzyme involved in H2S production in F. nucleatum. The reaction catalyzed by lanthionine synthase involved the condensation of two L-cysteine molecules to produce L,L-lanthionine and H2S. The production of lanthionine in F. nucleatum is a vital process because this bacterium incorporates meso-lanthionine into its peptidoglycan layer. Without this diamino acid, the integrity of the cell wall would be disrupted, thus causing death of the bacterium. The only known synthetic route to make lanthionine in F. nucleatum is through lanthionine synthase; however, the product of this reaction is the L,L-isomer. Therefore, we wanted to investigate possible physiological routes of meso-lanthionine production. The insertion of meso-lanthionine in the peptidoglycan layer is a unique property of F. nucleatum because other gram-negative bacteria instead contain meso-2,6-diaminopimelic acid (meso-DAP). The bacteria that incorporate meso-DAP into the cell wall encode a DAP epimerase to convert L,L-DAP to the meso-isomer. Therefore, we considered the possibility that F. nucleatum produces meso-lanthionine in the same manner. We investigated Fn1732 as a putative lanthionine epimerase due to it being the closest homolog to the gene encoding for DAP epimerase (dapF). However, given the low activity of the enzyme, it is likely that Fn1732 is not a lanthionine epimerase and instead uses its conserved cysteine residues to catalyze a racemization reaction on another amino acid. The most likely route for meso-lanthionine production is through the reaction of L-cysteine and D-cysteine, catalyzed by lanthionine synthase. HPLC analysis of this reaction showed that meso-lanthionine was produced preferentially in a 3:1 ratio compared to L,L-lanthionine. There is little information on the levels of D-cysteine within the cell, however, in F. nucleatum it may be produced through spontaneous racemization or by a serine racemase. Given that lanthionine synthase has a slightly relaxed substrate scope, we also wanted to explore the possibility of the enzyme being able to react with different cysteine derivatives to produce lanthionine analogs. Indeed, we were able to react D-cysteine, D-cysteine methyl ester, and L-cysteine methyl ester with the a-aminoacrylate intermediate. The production of lanthionine analogs could be useful in the synthesis of peptidoglycan fragments to further our knowledge of peptidoglycan structure, remodeling, and sensing. The ability of lanthionine synthase to produce these lanthionine analogs also prompted us to react the enzyme with other cysteine or DAP derivatives to find potential inhibitors. We determined that out of nine tested inhibitors, meso-DAP and meso-oxa-DAP have the most potential for inhibition of lanthionine synthase. Molecular modeling demonstrated that these meso-isomers exhibited strong binding due to the formation of 166 an additional hydrogen bond with S70 in the lanthionine synthase active site. Therefore, future research into the inhibition of lanthionine synthase should consider inhibitors with D-stereochemistry. Given that lanthionine synthase is required for F. nucleatum viability, this enzyme could be considered a viable drug target for the development of new antimicrobials. Chapter 5 focused on enzymes involved in the lysine fermentation pathway, a pathway that is vital to F. nucleatum since it relies on the fermentation of amino acids for growth and survival. Metabolism of lysine is also important for coaggregation of F. nucleatum with other bacteria because the accumulation of lysine inhibits the fusobacterial adhesin RadD. Lysine 5,6-aminomutase (5,6-LAM) is a PLP- and AdoCbl-dependent isomerase that catalyzes the second step of this pathway, the 1,2-rearrangement reaction of b-L-lysine to erythro-3,5-diaminohexanoate. During catalysis, 5,6-LAM undergoes substrate-induced inactivation where the enzyme is unable to participate in another round of catalysis due to the irreversible oxidation of the AdoCbl cofactor to hydroxocobalamin. This process is also a mechanistic feature of the AdoCbl-dependent mutases and eliminases; however, these enzymes have evolved to have metallochaperone systems in place that use ATP or GTP to restore the activity of the respective AdoCbl-dependent enzyme. An analogous chaperone system has not yet been identified for the aminomutases. Coincidentally, two genes within the F. nucleatum lysine fermentation operon that are downstream to those that encode for 5,6-LAM, kamB and kamC, have not been characterized. Our studies showed that these gene products form a complex as KamBC and function as a reactivating factor for 5,6-LAM by exchanging the inactivated cofactor for free AdoCbl in an ATP-dependent manner. Further research is needed to determine the exact mechanism by which KamBC exerts this activity and how it interacts with 5,6-LAM. 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Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Wolthers, Kirsten
Uncovering distinct aspects of amino acid metabolism in Fusobacterium nucleatum
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