Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Hoffarth, Elesha Rene
Doctor of Philosophy - PhD
University of British Columbia
Natural products are essential to the discovery of new drugs, including antibiotics. Industrial interest in natural products has declined since the 1980s, but advances in biocatalysis and biosynthetic knowledge have helped revive interest in natural products as these advances contribute to more feasible discovery, production and derivatization of natural products. To continue this industrial interest in natural products and their related compounds, work should be done to accumulate more biosynthetic knowledge to further improve methods of discovery, production and derivatization and facilitate more widespread use of biocatalysts. Indolmycin is a natural product with antibiotic activities against methicillin-resistant Staphylococcus aureus, Helicobacter pylori and Plasmodium falciparum, whose biosynthetic pathway is shown here to be a source of new biochemical tools. First, in order to better understand the unique reactivity of the rare oxygen- and pyridoxal 5′-phosphate (PLP)-dependent arginine desaturases discovered from the indolmycin biosynthetic pathway, the first X-ray crystal structure of an arginine desaturase was solved. This structure showed an active site that was highly similar to the related oxygen- and PLP-dependent hydroxylases. Catalytic residues for the arginine desaturases were uncovered by creating mutagenic variants based on the crystal structure information. Second, sequence similarity analysis and side-product analysis were done, which further supported a higher similarity to the arginine hydroxylases than was originally predicted. Additionally, superoxide was shown to be an intermediate of the arginine oxidase mechanism for the first time through EPR and cytochrome c assays. Based on this information, a unified mechanistic hypothesis is proposed which suggests that desaturation and hydroxylation may be differentiated by the presence/position of water in the active site. Third, the indolmycin biosynthetic enzymes are used in conjunction with a promiscuous tryptophan synthase and a three-step chemical synthesis to produce indolmycin and several novel halogenated derivatives. Derivatives with fluorinated indole substitutions showed a moderate bioactivity against S. aureus and could be useful in developing indolmycin for clinical use. Overall, this work uses the indolmycin biosynthetic enzymes to expand the known biocatalytic repertoire with the hope that it can contribute to more widespread use of biocatalysts in the production of natural product-derived molecules.
BUILDING CHEMICAL TOOLS FROM THE INDOLMYCIN BIOSYNTHETIC PATHWAY by ELESHA RENE HOFFARTH B.Sc., The University of Calgary, 2014 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2021 © Elesha Rene Hoffarth, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Building chemical tools from the indolmycin biosynthetic pathway. submitted by Elesha Rene Hoffarth in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Examining Committee: Dr. Katherine S. Ryan, Associate Professor, Chemistry, University of British Columbia Supervisor Dr. Stephen G. Withers, Professor, Chemistry, University of British Columbia Supervisory Committee Member Dr. Laurel L. Schafer, Professor, Chemistry, University of British Columbia University Examiner Dr. Natalie Strynadka, Professor, Biochemistry and Molecular Biology, University of British Columbia University Examiner Dr. Erika Plettner, Professor, Chemistry, Simon Fraser University External Examiner Additional Supervisory Committee Members: Dr. Mark MacLachlan, Professor, Chemistry, University of British Columbia Supervisory Committee Member Dr. Martin Tanner, Professor, Chemistry, University of British Columbia Supervisory Committee Member iii Abstract Natural products are essential to the discovery of new drugs, including antibiotics. Industrial interest in natural products has declined since the 1980s, but advances in biocatalysis and biosynthetic knowledge have helped revive interest in natural products as these advances contribute to more feasible discovery, production and derivatization of natural products. To continue this industrial interest in natural products and their related compounds, work should be done to accumulate more biosynthetic knowledge to further improve methods of discovery, production and derivatization and facilitate more widespread use of biocatalysts. Indolmycin is a natural product with antibiotic activities against methicillin-resistant Staphylococcus aureus, Helicobacter pylori and Plasmodium falciparum, whose biosynthetic pathway is shown here to be a source of new biochemical tools. First, in order to better understand the unique reactivity of the rare oxygen- and pyridoxal 5′-phosphate (PLP)-dependent arginine desaturases discovered from the indolmycin biosynthetic pathway, the first X-ray crystal structure of an arginine desaturase was solved. This structure showed an active site that was highly similar to the related oxygen- and PLP-dependent hydroxylases. Catalytic residues for the arginine desaturases were uncovered by creating mutagenic variants based on the crystal structure information. Second, sequence similarity analysis and side-product analysis were done, which further supported a higher similarity to the arginine hydroxylases than was originally predicted. Additionally, superoxide was shown to be an intermediate of the arginine oxidase mechanism for the first time through EPR and cytochrome c assays. Based on this information, a unified mechanistic hypothesis is proposed which suggests that desaturation and hydroxylation may be differentiated by the presence/position of water in the active site. Third, the indolmycin biosynthetic enzymes are used in conjunction with a promiscuous tryptophan synthase and a three-step chemical synthesis to produce indolmycin and several novel halogenated derivatives. Derivatives with fluorinated indole substitutions showed a moderate bioactivity against S. aureus and could be useful in developing indolmycin for clinical use. Overall, this work uses the indolmycin biosynthetic enzymes to expand the known biocatalytic repertoire with the hope that it can contribute to more widespread use of biocatalysts in the production of natural product-derived molecules. iv Lay Summary Natural products are molecules from natural sources, such as bacteria, fungi and plants. The discovery and production of natural products is essential for drug development, as nearly half of all approved drugs are derived from natural products. Nature produces these molecules using biosynthetic enzymes. Advances in genomics and knowledge of biosynthetic enzymes has allowed biosynthetic enzymes to be more frequently harnessed by companies to make useful natural products. Here, the biosynthetic enzymes used by nature to make indolmycin, a natural product, are studied for their unique characteristics and their ability to make non-natural, indolmycin-like molecules. This study expands the current knowledge of biosynthetic enzymes, which helps make industrial use of biosynthetic enzymes more accessible. v Preface Some parts of Chapter 1 were published as a review article: Hoffarth, E. R., Rothchild, K. W. & Ryan, K. S. Emergence of oxygen‐ and pyridoxal phosphate‐dependent reactions. FEBS J. 287, 1403–1428 (2020). E. R. H. (the author) wrote most of the sections based on the manuscript and all other section in Chapter 1. Sections 1.4.3.3, 1.4.4.1 and 1.4.4.2 were originally drafted by K. W. R. and parts of Section 1.3.1 were originally drafted by K. S. R (PhD supervisor). The manuscript and this chapter were also reviewed and edited by K. S. R. A version of Chapters 2 and 3, combined, has been submitted for publication: Hoffarth, E. R., Haaviet, K. C., Kuatsjah, E., MacNeil, G. A., Garcia-Borràs, M., Saroya, S., Walsby, C. J., Eltis, L. D., Houk, K. N., Ryan, K. S. A shared mechanistic pathway for pyridoxal phosphate-dependent arginine oxidases (in revision). E. R. H. wrote most of these chapters and completed most experiments and data analyses. E. R. H. and K. S. R designed this research project. Some of the Ind4 homolgs described in Chapters 2 and 3 were partially purified by S. S. In Chapter 2, Sections 2.2.5 and 2.3.1.3, the DFT calculations were completed by M. G.-B. and the methods describing these calculations in Section 2.2.5 were originally drafted by K. C. H, both under the supervision of K. N. H. Experiments and data analysis to determine the kinetic parameters of Plu4 described in Chapter 3, Sections 3.2.5 and 3.3.2.1 were completed by E. K under the supervision of L. D. E. Under the supervision of C. J. W., EPR data in Section 3.3.2.3 was collected and processed by G. A. M., who also wrote the EPR instrumentation methods in Section 3.2.6. The DFT calculations from the submitted manuscript that are briefly summarized in Section 3.3.3, were completed by K. C. H under the supervision of K. N. H. The manuscript and these chapters were reviewed and edited by K. S. R. A version of Chapter 4 has been submitted for publication: Hoffarth, E. R., Kong, S., He, H.-Y., Ryan, K. S. An engineered biosynthetic-synthetic platform for production of halogenated indolmycin antibiotics (in revision). E. R. H. wrote this chapter and completed most experiments and data analyses. E. R. H. and K. S. R designed this research project. H.-Y. H. helped design and set up the synthetic aspects of the project. S. K. and H.-Y. H. helped isolate some of the indolmycenic acid derivatives and completed synthetic steps for some of the indolmycin derivatives. NMR data was collected by staff at the NMR Facility in the Department of Chemistry at the University of British Columbia. HR-MS data was collected by staff at the University of British Columbia Mass Spectrometry Center. Disk diffusion and MIC bioactivity vi data was collected by Jessie Chen at the Biolocal Services Laboratory in the Department of Chemistry at the University of British Columbia. The manuscript and this chapter was reviewed and edited by K. S. R. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ...................................................................................................................xv Acknowledgements .................................................................................................................. xviii Chapter 1: Introduction ................................................................................................................1 1.1 Natural products .............................................................................................................. 1 1.1.1 A brief history and current perspective ............................................................... 1 1.1.2 Antibiotics ........................................................................................................... 3 1.1.3 The importance of biocatalysis in natural products ............................................ 4 1.2 Indolmycin ...................................................................................................................... 5 1.2.1 Bioactivity ........................................................................................................... 7 1.2.2 Synthesis ............................................................................................................. 9 1.2.3 Biosynthesis ...................................................................................................... 11 1.3 PLP-dependent enzymes ............................................................................................... 13 1.3.1 The cofactor and mechanism ............................................................................ 13 1.3.2 PLP-dependent enzymes as biocatalysts ........................................................... 16 1.3.2.1 Tryptophan synthase (TrpS) ............................................................................. 16 1.4 Oxygen- and PLP-dependent enzymes ......................................................................... 18 1.4.1 Understanding reactivity of PLP-dependent enzymes with O2 ......................... 21 1.4.2 Paracatalytic reactions of PLP-dependent enzymes with O2 ............................ 23 1.4.3 Oxidative deamination ...................................................................................... 27 1.4.3.1 Plant amino acid aldehyde synthases ................................................................ 27 1.4.3.2 Insect amino acid aldehyde synthases ............................................................... 29 1.4.3.3 Lincosamide biosynthetic enzyme CcbF .......................................................... 30 1.4.4 Decarboxylating oxygenation ........................................................................... 32 viii 1.4.4.1 PvdN ................................................................................................................. 32 1.4.4.2 Cap15 ................................................................................................................ 34 1.4.4.3 CuaB ................................................................................................................. 36 1.4.5 Arginine oxidation ............................................................................................ 37 1.4.5.1 MppP ................................................................................................................. 37 1.4.5.2 RohP .................................................................................................................. 39 1.4.5.3 Ind4 ................................................................................................................... 41 1.5 Aims of study ................................................................................................................ 42 Chapter 2: Crystallization and mutagenesis of arginine oxidases...........................................44 2.1 Introduction ................................................................................................................... 44 2.2 Materials and methods .................................................................................................. 46 2.2.1 General methods, materials and strains ............................................................ 46 2.2.2 Cloning and mutagenesis .................................................................................. 47 2.2.3 Protein purification and Plu4/Ind4 variant characterization ............................. 49 2.2.4 Crystallization ................................................................................................... 50 2.2.5 Data collection, structure determination and computational methods .............. 51 2.3 Results ........................................................................................................................... 55 2.3.1 Plu4 crystal structures ....................................................................................... 55 2.3.1.1 Holo-enzyme structure ...................................................................................... 56 2.3.1.2 Intermediate-bound structure ............................................................................ 60 2.3.1.3 Product-bound structure .................................................................................... 65 2.3.2 Site-directed mutagenesis ................................................................................. 69 2.4 Discussion ..................................................................................................................... 73 Chapter 3: Understanding the evolutionary relationship and bioactivity of arginine desaturases ....................................................................................................................................75 3.1 Introduction ................................................................................................................... 75 3.2 Materials and methods .................................................................................................. 76 3.2.1 General methods, materials and strains ............................................................ 76 3.2.2 Network analysis, sequence alignment, phylogenetic trees and homolog identification ......................................................................................................................... 77 3.2.3 Cloning .............................................................................................................. 78 ix 3.2.4 Protein purification ........................................................................................... 79 3.2.5 In vitro characterization .................................................................................... 79 3.2.6 Electron paramagnetic resonance (EPR) .......................................................... 81 3.3 Results ........................................................................................................................... 81 3.3.1 Sequence similarity networks ........................................................................... 81 3.3.2 In vitro desaturase characterization .................................................................. 86 3.3.2.1 Product analysis and kinetics ............................................................................ 86 3.3.2.2 Side-product analysis ........................................................................................ 90 3.3.2.3 Demonstrating the existence of superoxide ...................................................... 93 3.3.3 Density functional theory calculations .............................................................. 96 3.4 Discussion ..................................................................................................................... 96 Chapter 4: Engineered production of indolmycin derivatives through semi-synthesis ......102 4.1 Introduction ................................................................................................................. 102 4.2 Materials and methods ................................................................................................ 103 4.2.1 General methods, materials and strains .......................................................... 103 4.2.2 Cloning ............................................................................................................ 104 4.2.3 Expression and feeding ................................................................................... 106 4.2.4 Extraction and purification of indolmycenic acid and its derivatives ............. 107 4.2.5 Synthesis of indolmycin and indolmycin derivatives ..................................... 110 4.2.6 Bioactivity assays ............................................................................................ 115 4.3 Results ......................................................................................................................... 116 4.3.1 Production of indolmycin ............................................................................... 116 4.3.1.1 Heterologous expression of indolmycin genes ............................................... 116 4.3.1.2 Indolmycenic acid purification and indolmycin synthesis .............................. 118 4.3.2 Production of indolmycin derivatives ............................................................. 121 4.3.2.1 Use of TrpS for improved indole incorporation ............................................. 121 4.3.2.2 Scope of indole incorporation ......................................................................... 123 4.3.2.3 Production of indolmycin derivatives ............................................................. 126 4.3.3 Bioactivity against MRSA .............................................................................. 131 4.4 Discussion ................................................................................................................... 134 Chapter 5: Conclusions and future directions ........................................................................138 x 5.1 General Conclusions ................................................................................................... 138 5.2 Future Directions ........................................................................................................ 139 5.2.1 Arginine oxidases ............................................................................................ 139 5.2.2 Indolmycin ...................................................................................................... 142 References ...................................................................................................................................145 Appendices ..................................................................................................................................166 Appendix A: Supplementary information for Chapter 3 ........................................................ 166 Appendix B: Supplementary information for Chapter 4 ......................................................... 174 xi List of Tables Table 1.1: PLP-enzymes with oxygen-dependent or paracatalytic reactions ............................... 20 Table 2.1: Bacterial strains and plasmids used in this study ........................................................ 46 Table 2.2: Oligonucleotide primers used in this study ................................................................. 48 Table 2.3: Data collection and refinement statistics .................................................................... 53 Table 2.4: Cartesian coordinates of optimized structures ............................................................ 54 Table 2.5: Energies of imine and enamine tautomers .................................................................. 54 Table 2.6: RMSD values for Plu4 ................................................................................................ 57 Table 2.7: Product profiles of enzyme variants ............................................................................ 70 Table 3.1: Bacterial strains and plasmids used in this study ........................................................ 76 Table 3.2: Enzymes used in this study ......................................................................................... 78 Table 3.3: Oligonucleotide primers used in this study ................................................................. 79 Table 4.1: Strains and plasmids used in this study ..................................................................... 104 Table 4.2: Primers used in this study ......................................................................................... 105 Table 4.3: Amounts of 11-derivatives and 6-derivatives obtained ............................................ 121 Table 4.4: HR-MS analysis of purified 11, 6 and their derivatives ........................................... 131 Table 4.5: MIC50 values determined for 6 and its derivatives against MRSA ........................... 133 xii List of Figures Figure 1.1: Timeline of antibiotic discovery and resistance .......................................................... 2 Figure 1.2: TrpRS-binding molecules and the TrpRS mechanism ................................................ 6 Figure 1.3: Synthetic strategies towards the production of indolmycin ...................................... 10 Figure 1.4: Indolmycin derivatives that have been made synthetically. ...................................... 11 Figure 1.5: The indolmycin biosynthetic pathway ....................................................................... 13 Figure 1.6: Mechanisms of forming common quinonoid intermediates ...................................... 15 Figure 1.7: TrpS catalyzes the production of tryptophan ............................................................. 18 Figure 1.8: Proposed mechanism for paracatalytic oxidative deamination ................................. 22 Figure 1.9: Mechanisms of decarboxylation, abortive transamination and oxidative deamination in the paracatalytic decarboxylases ............................................................................................... 25 Figure 1.10: Overall schemes for the three types of PLP-dependent decarboxylases with paracatalytic reactivity .................................................................................................................. 26 Figure 1.11: X-ray crystal structure of human GAD67 ............................................................... 27 Figure 1.12: Schemes for physiological roles of major AAS enzymes ....................................... 28 Figure 1.13: Partial sequence alignments comparisons of AASs and AADCs ............................ 29 Figure 1.14: Biosynthesis of lincomycins and celesticetins ........................................................ 32 Figure 1.15: Pyoverdine and proposed mechanism of PvdN ....................................................... 34 Figure 1.16: Structures and biosynthesis of capuramycin-type molecules .................................. 35 Figure 1.17: Proposed mechanism of Cap15 ............................................................................... 36 Figure 1.18: The enduracididine biosynthetic pathway and gene cluster .................................... 38 Figure 1.19: Proposed mechanism for known arginine oxidase products ................................... 39 Figure 1.20: The azomycin gene cluster and biosynthetic pathway ............................................ 40 Figure 2.1: Scheme of O2- and PLP-dependent arginine oxidase reactivities ............................. 45 Figure 2.2: Plu4 crystals ............................................................................................................... 55 Figure 2.3: X-ray crystal structures of Plu4 ................................................................................. 56 Figure 2.4: Comparison of holo-enzyme overall structures ......................................................... 58 Figure 2.5: Plu4 Holo-enzyme active site analysis ...................................................................... 59 Figure 2.6: Comparison of possible intermediates ....................................................................... 61 Figure 2.7: Comparison of N-termini in intermediate-bound structures ..................................... 63 xiii Figure 2.8: Plu4 intermediate ligand interactions with protein in the intermediate-bound structure ......................................................................................................................................... 64 Figure 2.9: Comparison of Plu4, RohP (blue; PDB: 6C3D) and SwMppP (plum; 6C8T) intermediate-bound active site structures ...................................................................................... 65 Figure 2.10: DFT calculations for four electron oxidized product in imine and enamine forms. 66 Figure 2.11: Electron density for products 2 and 4 ...................................................................... 67 Figure 2.12: Analysis of product binding conformations ............................................................ 68 Figure 2.13: Comparison of Plu4, RohP (blue; PDB: 6C3A) and SwMppP (plum; 6C9B) product-bound active site structures ............................................................................................. 69 Figure 2.14: Alignment of sequences from characterized SSN groups ....................................... 70 Figure 2.15: Full sequence alignment of representative arginine oxidases ................................. 72 Figure 2.16: Electrostatic map of the Plu4 active site pocket surrounding Quinonoid I ............. 74 Figure 3.1: Sequence similarity network analysis for the arginine oxidases ............................... 84 Figure 3.2: Phylogenetic trees of the arginine oxidase family of enzymes ................................. 85 Figure 3.3: LC-MS assay of known and newly discovered arginine desaturases ........................ 87 Figure 3.4: Plu4 reaction products and kinetics ........................................................................... 88 Figure 3.5: Plu4 reaction products measured by LC-MS showing didehydroarginine ................ 89 Figure 3.6: Measurement of Plu4 pH optimum ........................................................................... 89 Figure 3.7: Desaturase side-product controls ............................................................................... 91 Figure 3.8: Plu4 assay with 50% H218O ....................................................................................... 91 Figure 3.9: Optimization of OPD derivatization conditions using SwMppP .............................. 92 Figure 3.10: Quantification of hydroxylated product comparing Plu4 and SwMppP ................. 93 Figure 3.11: EPR measurements demonstrating production of superoxide ................................. 94 Figure 3.12: EPR spectra of CMH-trapped superoxide during control reactions ........................ 95 Figure 3.13: Absorbance spectra of Plu4 reaction assayed in the presence of cytochrome c ...... 95 Figure 3.14: Reaction schemes for examples of non-isofunctional phylogenetic clustering ..... 100 Figure 3.15: Proposed mechanism of arginine desaturases and hydroxylases .......................... 101 Figure 4.1: Indolmycin gene cluster and biosynthetic pathway ................................................. 103 Figure 4.2: Synthetic scheme to 6 from 11 ................................................................................ 111 Figure 4.3: Predicted tautomeric isomers of indolmycin observed from 1H NMR spectra. The tautomer on the left is the natural tautomer. ............................................................................... 112 xiv Figure 4.4: Cloning genes for in vivo production of 6, 11 and derivatives of 11 ...................... 116 Figure 4.5: Summary of compound [M+H]+ ions. ..................................................................... 117 Figure 4.6: LC-MS analysis of observed products from E. coli I1234670P5 ............................ 118 Figure 4.7: HPLC comparison of E. coli I120 and E. coli I1234670P5 .................................... 119 Figure 4.8: LC-MS analysis for synthesis and purification of 6 ................................................ 120 Figure 4.9: UV-Vis traces obtained during LC-MS analysis of synthetic steps ........................ 121 Figure 4.10: TrpS addition to the biosynthetic platform ............................................................ 122 Figure 4.11: HPLC analysis of 5F-11 amounts under different feeding conditions .................. 123 Figure 4.12: LC-MS analysis of halogen-substituted indole incorporation ............................... 124 Figure 4.13: LC-MS analysis of hydroxyindole or azaindole incorporation ............................. 125 Figure 4.14: Semi-synthetic scheme of producing 6 and its derivatives ................................... 127 Figure 4.15: LC-MS analysis of 11-derivative esterification .................................................... 128 Figure 4.16: LC-MS analysis of 13-derivative oxazolinone-cyclizations ................................. 129 Figure 4.17: LC-MS analysis of 14-derivative N-methylation .................................................. 130 Figure 4.18: Disk diffusion assay of 6 and its derivatives against MRSA ................................ 132 Figure 4.19: Docking of indolmycin derivatives made .............................................................. 134 xv List of Abbreviations AADC: amino acid decarboxylase AARS: aminoacyl-tRNA synthetase AAS: amino acid aldehyde synthase AEE: ʟ-Ala-D/ʟ-Glu epimerases AMD: a-methylDOPA amd: a-methyldopa hypersensitive gene AMP: adenosine monophosphate AONS: 8-amino-7-oxononanoate synthase CarU: uridine 5′-carboxoamide CDC: Center for Disease Control and Prevention CMH: cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine DFT: density functional theory DHPAA: 3,4-dihydroxyphenylacetaldehyde DHPAAS: DHPAA synthase DMSO: dimethyl sulfoxide DOPA: 3,4-dihydroxyphenylalanine DDC: DOPA decarboxylase EFI-EST: Enzyme Function Initiative-Enzyme Similarity Tool EFI-GNT: Enzyme Function Initiative-Genomic Neighbourhood Tool EPR: electron paramagnetic resonance ESI-MS: electrospray ionization-mass spectrometry GAD: glutamate decarboxylase GlyU: 5′-glycyluridine G3P: glyceraldehyde-3-phosphate HPLC: high-performance liquid chromatography HR-MS: high resolution-mass spectrometry L-HTP: L-hydroxytryptophan HTS: high-throughput screening IDT: Integrated DNA Technologies xvi IPTG: isopropyl β-D-1-thiogalactopyranoside LB: Luria-Bertani TOF-MS: time of flight-mass spectrometer LC-MS: liquid chromatography-mass spectrometry MHB: Mueller-Hinton Broth MIC: minimum inhibitory concentration MRSA: methicillin-resistant Staphylococcus aureus NAAAR: N-acylamino acid racemase NADH: nicotinamide adenine dinucleotide NADPH: nicotinamide adenine dinucleotide phosphate NEB: New England Bio Labs NMR: nuclear magnetic resonance ODC: ornithine decarboxylase OPD: o-phenylenediamine OSBS: o-succinylbenzoate synthase PAAS: phenylacetaldehyde synthase PCR: polymerase chain reaction PHA: phenylacetaldehyde PLP: pyridoxal 5′-phosphate PMP: pyridoxamine 5′-phosphate PTPS: 6-pyruvoyl-tetrahydropterin synthases RMSD: root-mean-square deviation SAM: S-adenosylmethionine SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis SOD: superoxide dismutase SSN: sequence similarity network TLS: translation-liberation-screw TrpA: tryptophan synthase a-subunit TrpB: tryptophan synthase b-subunit TrpRS: tryptophanyl-tRNA synthetase TrpS: tryptophan synthase xvii UV-Vis: ultraviolet-visible xviii Acknowledgements I would like to thank my supervisor, Dr. Katherine Ryan, for constantly encouraging me throughout my graduate studies and being incredibly supportive of my career goals. She has taught me so much about the research process and I am constantly amazed at all of the expertise she had to share about communicating my work. Thank you to Melanie Higgins, Hai-Yan He, Yi-Ling Du and Lona Alkhalaf, who mentored me during my graduate studies and to all other Ryan lab members, past and present, that I have had the pleasure to work with. I would also like to thank my supervisory committee members for their encouragement and helpful suggestions to improve my thesis. Thank you to the many wonderful friends I have made during my time at the University of British Columbia, namely Elizabeth Fisher, Kelsi Lix, Phil Danby, Trevor Cross and Chloe Gerak, whom I am so grateful for. I am so appreciative of the staff in the Chemistry Department at UBC, including Elena Polishchuck and Jessie Chen in the Biological Services Laboratory for being extremely helpful because their hard work was essential to making many of my research tasks run smoothly. Maria Ezhova in the NMR Facility was so valuable in obtaining and interpreting my NMR spectra. Ben Herring in the Shared Instrument Facility was so friendly and accommodating of my abundant LC-MS needs. Thank you to Anne Thomas who was an amazing teaching assistant supervisor to me and I appreciate that she kept me on in teaching positions that I have enjoyed very much throughout these last few years. I have also enjoyed our many discussions about the best food in the West End of Vancouver. I am extremely grateful to my parents, Lisa and Lenard Hoffarth, who shaped my independence, confidence and curiosity that I have needed to complete my research. They have taught me how to work hard and persevere to achieve my goals and they have always supported my desire to further my education. Most of all, I would like to thank my fiancé, Jack, for his unwavering encouragement throughout my entire post-secondary and graduate education. He is especially appreciated for trying very hard to give me the quiet time that I needed to complete work and for occasionally picking me up from UBC when it was raining. He has comforted me through the hardest times and celebrated with me through the happiest moments.1 Chapter 1: Introduction1 To begin exploring the development of biochemical tools from the indolmycin biosynthetic pathway, this section will review relevant background information. First, the history of natural products will be described, followed by an analysis of natural products as antibiotics and the importance of biocatalysis in producing natural product-derived compounds. Then, indolmycin will be introduced with a focus on its discovery, bioactivity, synthesis and biosynthesis. Pyridoxal 5′-phosphate (PLP)-dependent enzymes are an essential part of indolmycin biosynthesis and will be briefly described with a focus on biocatalytic uses to introduce the unique and rare oxygen- and PLP-dependent enzymes. The scope and current mechanistic understanding of the oxygen- and PLP-dependent enzymes, including Ind4, an indolmycin biosynthetic enzyme, will be reviewed. Finally, the aims of this thesis will be described as they relate to use of the indolmycin biosynthetic pathway for building biochemical tools. 1.1 Natural products 1.1.1 A brief history and current perspective Natural products are a diverse collection of molecules derived from natural sources, typically from the secondary metabolism of plants, bacteria, fungi and marine organisms.1,2 Secondary metabolites provide their producing organisms with a survival advantage in certain environments, but are not required for the organism’s survival under all circumstances.1,2 These molecules have been widely useful, not only to the producing organisms, but also for industrial, medicinal and scientific purposes because of their diverse properties, ranging from anticancer activity to phytotoxicity. Antibiotic development in particular has greatly benefited from the discovery of new natural products.3,4 Some of the earliest examples of human use of natural products come from ancient Egypt and Mesopotamia and were primarily from plant sources.2,5 Notable examples of natural products discovered from plants and developed for pharmaceutical use are salicin, from the willow tree (Salix alba L.), morphine from the opium poppy (Papaver somniferum L.) and artemisinin from Artemisia annua L., which all have roots in ancient 1 Parts of this chapter have been published: Hoffarth, E. R., Rothchild, K. W. & Ryan, K. S. Emergence of oxygen‐ and pyridoxal phosphate‐dependent reactions. FEBS J. 287, 1403–1428 (2020). 2 medicine.2,6 Evidence of microbial sources of disease-preventing therapies can be seen for thousands of years in the use of medicinal soils and mouldy bread to treat open wounds.7 However, research into microbial sources of natural products were not of particular focus until the discovery of penicillin by Fleming in 1928, which spurred the “Golden Era” of antibiotic discovery and the discovery of other microbial natural products as a result (Figure 1.1).2,5 By the 1990s, the “Golden Era” had ended as novel structures were being discovered less frequently. Natural product discoveries from pharmaceutical companies were being discontinued in favour of synthetic libraries due to incompatibility of the natural product libraries with high-throughput screening (HTS) methods that were becoming popular.8 The pharmaceutical environment and the blockbuster model of drug development necessitates accurate, quick and profitable projects via HTS as a result of pressure from shareholder expectations following success in the 1990s and early 2000s. The high structural complexity and high probability of redundant characterization are just some of the reasons natural product research has been largely discontinued.8 However, natural chemical diversity is unmatched in its ability to produce leads and inspire new compounds with close to half of all small molecule drugs approved between 1981-2014 being natural products or related molecules.2,9–11 More recently, advances in genomics, screening compatibility, analytical techniques, and biosynthetic knowledge have all made natural product development more feasible, which could potentially lead to a new revolution in natural product discovery.1,2,6,8,12 1930 1940 1950 1960 1970 1980 2010 20201990 2000Increasing ResistanceIndolmycin isdiscoveredGolden EraMajority of antibioticclasses discoveredOxazolidinonesare discoveredNew usesfor indolmycinare exploredActivity against MRSA, H. pylori,and P. falciparumPenicillin isdiscoveredFigure 1.1: Timeline of antibiotic discovery and resistance beginning with penicillin discovery in 1928, through to 2020. Timeline is adapted from previous review articles.7,15,16,18 3 1.1.2 Antibiotics Natural product research has been imperative to the discovery of new antibiotics. While examples of both natural and synthetic antibiotics exist, the importance of natural product discovery in the success of antibiotic development is not trivial.13,14 The first natural product-based, and possibly most famous, antibiotic discovered was penicillin. In 1928, Fleming classically observed inhibited bacterial growth around a contaminating fungus in a petri dish. This inhibited growth was later shown to be the result of the antibiotic penicillin, whose beta-lactam structure was solved by Dorothy Hodgkin in 1945.7,15,16 The discovery of penicillin inspired the exploration of many new antibiotics and other natural products during a period often referred to as the ‘Golden Era’ of antibiotic discovery (Figure 1.1), which ultimately saved countless lives and revolutionized modern medicine.7,15,16 However, the initial rapid discovery during the “Golden Era” resulted in misuse of antibiotics, which caused a quick rise in antibiotic resistance.7 Antibiotic resistance is an ancient mechanism bacteria have evolved to defend themselves against their own and other antimicrobials.15,17 Some antimicrobial resistance methods that bacteria have evolved include inactivation by an enzyme, efflux, mutations to decrease binding efficiency to the target, overproduction of the target, and decreased uptake.16,18 Phylogenetic studies of some b-lactamases, the enzyme responsible for inactivating b-lactam antibiotics like penicillin, revealed that they may have originated more than two billion years ago.17 Penicillinase, the enzyme responsible for inactivating penicillin in resistant strains, was discovered before penicillin was even put into clinical use.16 While antibiotic resistance mechanisms existed long before human use of antibiotics, it is undeniable that human use has increased the environmental presence of antibiotic resistance through use in medicine, agriculture, aquaculture, and waste disposal.16 Therefore, antimicrobial resistance is an inevitable consequence of antibiotic use that must be effectively managed.16,18 It is predicted that millions of people will die each year from antibiotic-resistant infections within a few decades if the issue is not dealt with.7,15 Unfortunately, the antibiotic resistance problem has been developing concurrently with dwindling antibiotic approval, ultimately exacerbating the effects of resistance.19 Reduced development of natural products following the “Golden Era” of antibiotics also resulted in reduced antibiotic discovery.7 The last new antibiotic classes discovered were the lipopeptides 4 with daptomycin in 1986 and the oxazolidinones with linezolid in 1987.15 However advances to natural product discovery has translated to new antibiotic discoveries.4,7,12,14,15,17,18 One method to find compounds helpful against antibiotic-resistant strains has been to revisit forgotten molecules that were originally discovered during the “Golden Era”, as some have shown remarkable activity against antibiotic-resistant bacteria.14–16 These new discoveries are a promising start to finding an abundance of new antibiotics and may signify a new era of antibiotic discovery to help overcome challenges with antibiotic resistance. However, overcoming these complex challenges will require cooperation from many groups of people to achieve success, including scientists, healthcare workers, the pharmaceutical and agricultural industries, policy makers, legislative bodies, and the general public.17 1.1.3 The importance of biocatalysis in natural products Advances in biosynthetic and biocatalytic knowledge have contributed to reinvigorated interest in natural products because of the powerful potential for this knowledge to aid in finding, producing and diversifying natural products more easily than extraction from natural producers or chemical methods alone.6,8,20 Due to advances in genomics, knowledge and characterization of existing biosynthetic pathways can lead to the prediction and discovery of novel natural products based on known biosynthetic scaffolds.3 New molecules with known pharmacophores can be predicted from genomic data once the biosynthetic genes responsible for building the pharmacophore are known. Investigation of species containing these biosynthetic genes would lead to more targeted searches for specific molecules with a desirable pharmocophore.1 Additionally, expression of silent, or “cryptic”, pathways has led to discoveries of new natural products that may have otherwise gone unnoticed without genomics and biosynthetic studies.6 However, many natural products cannot be predicted from their biosynthetic pathway and each pathway may need to be identified and characterized on an individual basis to understand the biomolecules they are making, especially for the most novel of cases.1 Methods of producing natural product-based compounds derived from biosynthetic knowledge include heterologous expression and metabolic engineering, among others,3,5,21,22 while methods of diversification include precursor feeding, synthetic biology, enzyme engineering and semi-synthesis.3,4,21,23–25 A key example of a natural product-derived compound produced though biocatalytic means was the recent biocatalytic cascade created by Merck for the 5 production of a potential HIV treatment, islatravir.26 Islatravir is a nucleoside analog with an alkynyl-substitution at the C4 position of 2-deoxyribose ring and a fluorine-substitution on the nucleobase. The biocatalytic cascade used to produce islatravir involved the use of nine enzymes, five of which were engineered for optimal performance, including tolerance of non-natural substrates. Compared to previous synthetic methods, this biocatalytic cascade showed improved atom economy and a reduction in the number of steps. Despite success with islatravir, major hurdles to more widespread biosynthetic production of natural product and their derivatives still exist. A few of these hurdles surround unannotated pathways and enzymes, incomplete knowledge of pathways and their enzymatic transformations and difficulty finding an enzyme to catalyze a desired reaction.22 Additionally, attempts to engineer individual enzymes to suit a desired reaction involve an in-depth knowledge of the enzymatic chemistry and may require a crystal structure.27 Overall, advances to biosynthetic and biocatalytic knowledge may help reinvigorate industrial interest in natural product development by aiding in the discovery, production and diversification of natural products. 1.2 Indolmycin Indolmycin is a tryptophan-derived antibiotic originally discovered from Streptomyces griseus ATCC12648 in 196028 (Figure 1.1) that acts as a competitive inhibitor of prokaryotic tryptophanyl-tRNA synthetase (TrpRS) based on structural similarity to tryptophan (Figure 1.2).29 It contains an oxazolinone ring connected to an indole through a chiral methine. A search for L-tryptophan antimetabolites in S. griseus identified indolmycin and N-desmethyl-C-desmethylindolmycin as L-tryptophan antagonists.30 N-desmethyl and C-desmethylindolmycin, along with stereoisomers of indolmycin, all show reduced bioactivity when compared to indolmycin (Figure 1.2).31 Although originally patented by Pfizer, indolmycin was not developed into a clinically used pharmaceutical because it had a narrow spectrum of activity and potential liver toxicity.32–37 However, challenges that arose in antimicrobial resistance since the “Golden Era” of antibiotic discovery have reinvigorated interest in indolmycin (Figure 1.1).32 Indolmycin was more recently shown to be active against methicillin-resistant Staphylococcus aureus (MRSA),38 Helicobacter pylori32 and Plasmodium falciparum.39 Revived interest in indolmycin even resulted in it being patented again by Takeda Pharmaceutical Company Ltd. for the treatment of H. pylori infections along with a method of production.40–43 However, due to the 6 lack of early interest in indolmycin, very few attempts have been made to improve the production of indolmycin and the derivatives that would potentially be needed for pharmaceutical development. Therefore, costs of purchasing the compound for research purposes have been kept very high at $185 USD per mg (Toronto Research Chemicals), as of July 2020, and no derivatives of the molecule are available for purchase. NHNH2O OHL-tryptophanNHOONNHindolmycinNHOONNH2N-desmethylindolmycinNHOONNHC-desmethylindolmycinNHOONNH2N-desmethyl-C-desmethylindolmycinNHSO OHchuangxinmycinabNHNH2O OHL-tryptophanATP PPiTrpRSNHNH2O OAMPAMPtRNATrpRSNHNH2O Ocharged tRNAFigure 1.2: TrpRS-binding molecules and the TrpRS mechanism. (a) Structures of tryptophan, a TrpRS substrate, and TrpRS inhibitors, indolmycin and chuangxinmycin.45,47 Indolmycin analogues with reduced bioactivity are also shown.30,31 (b) The process of making charged tRNA using TrpRS is shown. 7 1.2.1 Bioactivity TrpRS is a type of aminoacyl-tRNA synthetase (AARS) responsible for covalently attaching tryptophan to its specific tRNA molecule for further use in protein translation.44,45 The process occurs in two steps beginning with adenylation of tryptophan and release of pyrophosphate. Then, a covalent bond is formed between the tryptophan-specific tRNA and tryptophan, releasing adenosine monophosphate (AMP) and producing a “charged tRNA” molecule (Figure 1.2). The essential role of AARSs in protein translation makes them attractive drug targets against pathogens, as therapies targeting protein translation are commonly used as antimicrobials.14,44 Additionally, evolutionary differences between prokaryotic and eukaryotic AARSs allow these potential drugs to target pathogenic enzymes specifically.45,46 Mupirocin is the only AARS inhibitor currently in clinical use, in which it is used against S. aureus infections by targeting the S. aureus isoleucine-tRNA synthetase.44,46,47 Indolmycin and chuangxinmycin are both competitive inhibitors of TrpRS and mimic the tryptophan substrate (Figure 1.2).45,47 The biosynthetic gene clusters for both indolmycin and chuangxinmycin were discovered by searching producer genomes for colocalized TrpRS encoding genes that could confer resistance in the organisms, which may be an effective strategy to identify other biosynthetic gene clusters for AARS-targeting molecules.48,49 Initial interest in indolmycin began after it showed activity against a variety of bacteria.28,31 Tests on Escherichia coli cell lysates revealed that indolmycin targets prokaryotic TrpRS more specifically than mammalian TrpRS from calf and rat liver.29 It was later shown that indolmycin can inhibit the E. coli TrpRS with an IC50 of 9.25 nM and the H. pylori TrpRS at 12.2 nM compared to 4.04 mM for TrpRS from bovine liver, making it highly selective for the prokaryotic enzyme.32 Despite this specific inhibition of the prokaryotic TrpRSs, the binding affinity of tryptophan to both the eukaryotic and prokaryotic targets was similar (KM = ~2 µM), which led researchers to discover that inhibition of the bacterial TrpRS was mechanism-based.50 Through structural analysis of the Bacillus stearothermophilus TrpRS with indolmycin bound to the active site, it was found that indolmycin stabilized the Mg2+ and ATP complex in the prokaryotic TrpRS, resulting in reaction inhibition.50 Indolmycin was also shown to be a competitive inhibitor29 and was effective against sepsis caused by an antibiotic-resistant strain of Staphylococcus in mice.31 Two types of resistance towards indolmycin have been observed: high-level resistance as the result of an H43N mutation of TrpRS and low-level resistance 8 potentially from mutations to the uptake system.38 However, it also may also be possible for high-level resistance to occur due to horizontal gene transfer of the gene encoding a resistant TrpRS from a resistant strain.36 Despite showing inhibitory qualities against the E. coli TrpRS, indolmycin showed low levels of activity against gram negative bacteria and variable activity against many gram positive bacteria, making it a narrow spectrum antibiotic.32–36 The low activity against gram negative bacteria is thought to be due to an inability to cross the hydrophilic outer lipopolysaccharide membrane to reach the amino acid transport systems, as indolmycin was calculated to be 84.2% more lipophilic than tryptophan.33 Additionally, lower activities observed in gram positive bacteria have been attributed to differences in aromatic amino acid uptake mechanisms between different species.33 A narrow spectrum of activity was not the only reason that development of indolmycin was originally discontinued. Indolmycin was shown to be a potent inhibitor of tryptophan catabolism enzymes, tryptophan pyrrolase and tryptophan decarboxylase in rat livers, leading to potential liver toxicity.34,36,37 C-desmethylindolmycin, although weaker antimicrobially, demonstrated less of an effect on the eukaryotic system, demonstrating the importance of exploring derivative compounds in the future.37 More recent interest in indolmycin has come from exploration of new antibiotic uses for the compound (Figure 1.1). First, indolmycin was found to be a potent and selective bactericidal agent against H. pylori in 2001, showing stable activity even at low pH conditions that inactivated other anti-H. pylori compounds.32 Additionally, an in vivo experiment with Mongolian gerbils showed that indolmycin was effective at clearing H. pylori infections and repeated exposure of indolmycin to H. pylori, in vitro, did not produce any resistant strains.32 Indolmycin’s narrow spectrum of activity makes it an attractive treatment against H. pylori because it would be less disruptive to the gut microbiome, potentially avoiding unwanted gastrointestinal side effects of oral use.32 These attractive qualities lead to indolmycin and other oxazolinone derivatives being patented for use against H. pylori.36,40,41 Then, indolmycin’s activity against S. aureus was revisited because of ongoing challenges treating antibiotic resistant infections.38 Indolmycin was compared to current topical treatments for MRSA, mupirocin and fusidic acid, to which there is growing resistance threatening to disable the use of these treatments. Indolmycin was shown to have bacteriostatic activity against S. aureus that was similar to mupirocin and fusidic acid, but indolmycin was also 9 active against strains that were resistant to mupirocin and fusidic acid. In comparison of the bactericidal activity against H. pylori, it has been suggested that there are alternative modes of action for indolmycin in H. pylori that are not present in S. aureus. Analysis of resistance-causing mutations revealed that they arose less frequently to indolmycin than they did for mupirocin or fusidic acid. Additionally, high-level resistance to indolmycin, caused by an H43N mutation to the S. aureus TrpRS, disrupted the bacterial fitness of S. aureus with increases to doubling time and decreases to competitive fitness. Indolmycin was proposed as a topical agent against S. aureus so that resistance to the antibiotic would not interfere with its potential oral use in the treatment of H. pylori.36,38 Most recently, in 2016, indolmycin was shown to have bioactivity against P. falciparum.39 P. falciparum is a malaria-causing parasite that relies on protein synthesis to facilitate rapid growth. The apicoplast is a prokaryote-resembling organelle within P. falciparum where some of the protein translation machinery can be found within the organism. Indolmycin was found to inhibit the apicoplast TrpRS, specifically, causing parasite death in culture, making it potentially useful as a treatment against malaria.39 Indolmycin was also recently used to understand persistence in Chlamydia by mimicking tryptophan starvation.51 Bacterial persistence complicates treatment of infections and has been implicated in the evolution of antibiotic resistant bacteria,52 demonstrating that indolmycin could have uses in the development of antibiotics other than as a direct treatment of infections through use as a tool to study persistence and potential treatments against bacteria in this state. 1.2.2 Synthesis Several attempts have been made to make indolmycin and some of its derivatives since its discovery. Two main strategies to make indolmycin came from either directly linking the oxazolinone ring to the indole53,54 or by synthesizing an ester of indolmycenic acid, followed by cyclization of the oxazolinone ring (Figure 1.3). The latter was the more commonly applied strategy. There were three overall methods developed to make the indolmycenic acid ester. One method involved an epoxide ring opening through use of stannic chloride or with indolyl magnesium bromide.55–58 The epoxide could be made stereoselectively through lipase-assisted enantioselective deacetylation to obtain a chiral precursor to the epoxide.57 The second method of obtaining an indolmycenic acid ester was through installation of a chiral amine via a chiral 10 auxiliary-assisted Grignard reaction followed by oxygenation.59 The final and most recent method to obtain the indolmycenic acid ester was through a palladium catalyzed coupling of o-iodoaniline with an alkyne,60 analogous to Larock indole synthesis.61 The alkyne containing the correct stereocenters was obtained by lipase-assisted enantioselective acetylation.60 From the indolmycenic acid ester, there were three strategies to form the oxazolinone ring and obtain indolmycin. One was through reaction with N,N′-dimethylguanidine, but this method resulted in epimerization of the oxazolinone ring.31,55 The second method was to use N-methylthiourea59 and the third was to use guanidine with a subsequent amine exchange step.58,60 OOROindole and CH3MgBr or SnCl4NHOHOHOHClEtOH(esterification)NHOHOONHONONH240% MeNH2/H2ONHONONHOOOOTMS NHBocIPd(Ac)2,deprotection,deacetylationRlipase-assistedenantioselectiveacetylationOHOOTMSOArOROOlipase-assistedenantioselectivedeacetylationOOArOROOHOArOROOOOArOROOONHNHNH* NHOHHNONHNH2SSHNH2N NH2NHNHOORNHO HGrignard reactionwithchiral auxiliaryoxygenationNONONRCl*indolmycenicacidindolmycinFigure 1.3: Synthetic strategies towards the production of indolmycin.31,53–60 Strategies that resulted in diastereomers and/or epimers of the oxazolinone ring are indicated with an asterisk. 11 Several derivatives of indolmycin have been made by synthetic methods (Figure 1.4), including oxazolidinedione,31,58 thiazolinone62,63 and thiazolidinone63 analogs, all of which were modifications to the oxazolinone ring. An indolmycin derivative was also made with an additional ring to restrain movement of the molecule in the hopes that it would improve bioactivity.64 N- and C-desmethyl derivatives of indolmycin have been made31 along with some indole substituted analogs.58,60 Many of the syntheses of substituted indole analogs were focused on making known metabolites found from oral administration of indolmycin to rats, rather than for diversification towards pharmaceutical development.58 The types and locations of these substituents are also relatively narrow in scope, with attachments all being oxygen-based (hydroxy, methoxy, sulfoxy, and glucuronide) and only made at the 5- and 6-positions of the indole ring.58,60 1.2.3 Biosynthesis Early work on indolmycin biosynthesis done by Hornemann et al. (1971) established that indolmycin was derived from arginine, tryptophan and methionine.65 Further work partially purifying a tryptophan transaminase and C-methyltransferase revealed that these enzymes catalyze some of the initial steps of indolmycin production.66 An early precursor feeding experiment was also successful at obtaining 5-hydroxy and 5-methoxyindolmycin using the NHONONH2NHONONHNHONONHNHONHOONHSNHONHNHSNONHNHONONHHONHONONHONHONONHHO3SONHONONHGlucOC-desmethylindolmycin N-desmethylindolmycin oxazolidinedione derivative methoxy derivativehydroxy derivative glucuronide derivative sulfoxy derivativethiazolinone derivative thiazolidinone derivative ring fused derivativeFigure 1.4: Indolmycin derivatives that have been made synthetically. 12 respective tryptophan and indole derivatives with S. griseus ATCC 12648, resulting in improved bioactivity.67 The indolmycin biosynthetic gene cluster from S. griseus ATCC 12648 was previously elucidated by the Ryan group.48 Putative gene clusters were found by scanning the genome for genes that could potentially encode an indolmycin-resistant TrpRS and the gene cluster was confirmed through deletion mutants and in vitro characterization. It was found that indolmycin biosynthesis occurs in three main steps, consisting of biosynthesis of indolmycenic acid from tryptophan, dehydroarginine from arginine, and the oxazolinone ring from indolmycenic acid and dehydroarginine (Figure 1.5). This biosynthetic pathway will be discussed in further detail in Chapter 4. More recently, another indolmycin biosynthetic gene cluster was discovered from Pseudoalteromonas luteoviolacea, which appears to have arisen through convergent evolution.68 These indolmycin pathways differ by their assembly of the oxazolinone ring, in which the P. luteoviolacea biosynthetic method uses an evolved vicinal oxygen chelate superfamily enzyme to intercept a shunt product from the S. griseus pathway. 13 1.3 PLP-dependent enzymes 1.3.1 The cofactor and mechanism The indolmycin biosynthetic pathway includes two different PLP-dependent transformations, one of which, is an unprecedented oxygen-dependent desaturation of arginine catalyzed by Ind4, priming the arginine for reaction with adenylated indolmycenic acid. In order to better understand the importance of this unique enzyme, it is necessary to first understand PLP-dependent enzymes. PLP is a versatile organic cofactor widely employed in metabolism. The cofactor is the active form of vitamin B6, and it enables reactions with amino acid, oxoacid or amine substrates. The structure of PLP dictates several features about how it is used in enzymes. For example, its aldehyde functional group allows it to form a Schiff base with lysine NADHNAD+SAMSAHInd6NHH2N NH NH2OHONHH2N NH NHOHONHH2N NH NH2OHOInd4Ind5NADPHNHOHOOHInd3NHOHONH2Ind1Ind2NHNOO NHInd7ATaseInd62 O22 H2O2NADP+ATPAMPSAMSAHind3 ind6ind4 ind5 ind7ind2ind1ind0 ind81 kbFigure 1.5: The indolmycin biosynthetic pathway. The pathway shows that indolmycin derives from ʟ-arginine and ʟ-tryptophan, which are each tailored by indolmycin biosynthetic enzymes before cyclization and methylation to form indolmycin. Ind4, an O2- and PLP-dependent enzyme, converts ʟ-arginine to 4,5-dehydro-2-iminoarginine during indolmycin biosynthesis. 14 in the active site of PLP-dependent enzymes, giving the internal aldimine, which is the shared feature of most PLP-dependent enzymes and the starting point for catalysis (Figure 1.6). Furthermore, the pyridine ring enables PLP to stabilize carbanions through resonance. Using PLP, enzymes catalyze diverse reactions, ranging from transaminations to Claisen condensations; α-, β-, and γ-eliminations and replacements to transaldolations. The type of reaction catalyzed is dependent on the stereochemistry of the external aldimine intermediate and on which of the three a-carbon groups is aligned perpendicular with the conjugated ring system, as is described in Dunathan’s hypothesis.69 There are five main structural fold-types used to describe PLP-dependent enzymes; Fold-Type I (aspartate aminotransferase family), Fold-Type II (tryptophan synthase family), Fold-Type III (alanine racemase family), Fold-Type IV (D-amino acid aminotransferase family) and Fold-Type V (glycogen phosphorylase family).70 Some PLP-dependent enzymes have more recently been categorized into two additional fold-types.71 Despite their family names, each fold-type encompasses enzymes with many different reaction types and functions. PLP-dependent enzymes are estimated to account for ~4% of all enzyme activities, and recent chemo-proteomic strategies have enabled identification of unannotated proteins as PLP-dependent.72 Recently, some of the most common reactions that use PLP as a key cofactor in natural products biosynthesis were reviewed by the Ryan group, where the diversity of catalysis is on full display.73 Expansion of this enzymatic diversity through discovery and engineering will help build upon the PLP-dependent enzymes’ already impressive utilization as a biocatalyst. 15 NHNOORNH3OH2-O3POLysNHNOOROH2-O3PONH3LysExternalAldimineInternalAldimineNHNOOROH2-O3PONHNHOH2-O3PONHNHOH2-O3POH O OR ROOHNHNOOROH2-O3PONHNHOH2-O3PORNHNHOH2-O3POOOH+ CO2 RTransamination andRacemizationDecarboxylation ⍺-eliminationQuinonoidIntermediatesC⍺C4’C⍺C4’C⍺C4’C⍺C4’C⍺C4’ Figure 1.6: Mechanisms of forming common quinonoid intermediates in PLP-dependent enzymes based on Dunathan’s hypothesis, in which the leaving group determines the type of quinonoid intermediate formed. Each quinonoid intermediate is associated with certain types of reactions, such as transamination, racemization, decarboxylation and a-elimination. 16 1.3.2 PLP-dependent enzymes as biocatalysts The vast reactivity catalyzed by PLP-dependent enzymes in such stereo and regiospecific manners is an attractive quality for industrial processes of altering industrially important molecules, such as amino acids and other related amine-containing molecules.71 There have been several areas of potential PLP-dependent enzyme use previously outlined, including the chemical, pharmaceutical and food industries. Transaminases in particular have attracted the attention of organic chemists for their ability to produce chiral amines.74,75 Two notable examples include the use of an engineered transaminase in the production of an antidiabetic drug, sitagliptin,76 and an anxiety drug candidate precursor, imagabalin,77 with the former resulting in drastic improvements to yield, productivity, waste, heavy metal use and manufacturing cost.71 Other types of PLP-enzymes that may become industrially important include lysine decarboxylases, threonine aldolase, tyrosine phenyl-lyase, amino-caprolactam racemases, cystathionine b-lyase.71 Although these enzymes are all attractive for use in industry, more research and information is needed on these enzymes to make them amenable for industrial use. In order to be employed as a biocatalyst, the mechanistic details of each individual enzyme, even subtle differences, must be properly studied in order to fully understand the factors affecting catalysis.71 Going forward, the use of PLP-dependent enzymes for industrial processes will require discovery of new enzymes, mechanistic studies of current enzymes and engineering efforts to adapt these enzymes to suit the needs of industry. 1.3.2.1 Tryptophan synthase (TrpS) Although not yet used industrially, tryptophan synthase (TrpS) is a promising biocatalyst for the production of tryptophan derivatives. TrpS is a highly studied enzyme in the tryptophan biosynthesis pathway, converting indole-3-glycerol phosphate into tryptophan (Figure 1.7). The enzyme is heterodimeric with two dissimilar active sites, one per subunit.78,79 The a-subunit (TrpA) catalyzes a retro-aldol cleavage of indole-3-glycerol phosphate into indole and glyceraldehyde-3-phosphate (G3P). The b-subunit (TrpB) catalyzes a PLP-dependent condensation of indole and L-serine to form L-tryptophan via a b-replacement reaction. TrpS is the first enzyme that was found to allow internal passage of an intermediate between different active sites via a tunnel. While this indicates that indole is not released after the reaction in TrpA, it is possible to produce tryptophan by feeding indole and serine, thereby bypassing the activity 17 of TrpA and only employing TrpB.79 However, the overall activity of TrpB is reliant on the presence of TrpA, indicating some allosteric effects on regulation.80 Engineering efforts have been made to TrpB to negate any allosteric effects and allow it to act as a standalone enzyme.81–84 These efforts followed the discovery that TrpS, specifically the activity of TrpB, could not only produce tryptophan but a suite of tryptophan analogs when fed indole derivatives, making it an attractive biocatalyst.85–89 Substantial efforts to expand the substrate scope have been made to increase the utility of TrpS as a biocatalyst, including improved acceptance of indole-substituted and non-indole analogs, as well as L-serine derivatives, such as L-theronine.79 Recognition of the biocatalytic potential of TrpS has inspired biosynthetic researchers to include TrpS in several biocatalytic cascades to produce challenging molecules, such as D-tryptophan,90,91 tryptamine,92,93 psilocybin93 and thaxtomin94 derivatives. 18 1.4 Oxygen- and PLP-dependent enzymes As was previously discussed, a key necessity for more widespread use of biocatalysts in industrial processes is the expansion of the known biocatalytic repertoire. While PLP-dependent enzymes are already known to catalyze extremely diverse types of reactions, one exciting new reaction type being investigated amongst PLP-dependent enzymes is oxygen utilization. Despite NHHOOHOPO32-indole-3-glycerolphosphatetryptophansynthase(༾-subunit)NHindoletryptophansynthase(˟-subunit)tryptophanNHNH3OOG3P HOHO2-O3POL-serineOHONH2HOH2ONOHNLys2-O3PONONH2-O3POOOHO HNH2LysNONH2-O3POOOHONONH2-O3POOOHNNH2LysNONH2-O3POOONHHNH3LysNONH2-O3POOONHNONH2-O3POOONHL-serineH2OindoleL-tryptophanabFigure 1.7: TrpS catalyzes the production of tryptophan. (a) Overall reaction scheme for both the a-subunit and b-subunit in TrpS. (b) Catalytic cycle of TrpB producing L-tryptophan from L-serine and indole. 19 the reaction diversity amongst PLP-dependent enzymes, what was not originally expected is that these enzymes would use oxygen as a co-substrate during catalysis. Oxygen is more stable in the triplet state, necessitating a cofactor that can facilitate single-electron transfers. For this reason, it is more expected that enzymes that use oxygen would employ transition metal-containing cofactors and organic cofactors, like flavin, capable of such single electron transfers. Nonetheless, in pioneering papers, Schloss and Abell anticipated that any enzymatically generated carbanionic intermediates could react with electrophiles, including oxygen;95 a phenomenon often referred to as paracatalysis.96–98 They explored this hypothesis to identify a number of enzymes that react paracatalytically with oxygen, beautifully demonstrating that some (but not all) enzymes that stabilize carbanions can also react with oxygen. In recent years, the possibility that PLP-dependent enzymes can react with oxygen has been extended through discoveries in biosynthesis, highlighting enzymes that do not react paracatalytically with oxygen, but instead use oxygen as a dedicated co-substrate for the primary reaction of the enzyme. Described here, is the development of the hypothesis that PLP-dependent enzymes can react with oxygen and the experimental exploration of this hypothesis. Then recent work exploring the emergence of enzymes that use O2 as a co-substrate is described and the biological contexts of these enzymes are highlighted. It is interesting to note that O2-, PLP-dependent reactions have now been described in diverse protein families (Table 1.1), suggesting that the evolution of reactions with O2 may have emerged multiple times. This observation suggests that PLP-dependent enzymes that do not yet react with oxygen might also be evolved in the laboratory to use oxygen as a co-substrate. 20 Table 1.1: PLP-enzymes with oxygen-dependent or paracatalytic reactions. Table was adapted from Hoffarth et al. (2020). Enzyme Protein Family* Predicted Function* Reaction Catalyzed References DDC (paracatalytic) PF00282 DOPA decarboxylase Decarboxylation/Oxidative Deamination/Abortive Transamination Bertoldi et al. (1998)99 GAD (paracatalytic) PF00282 Glutamate Decarboxylase Decarboxylation/Oxidative Deamination/Abortive Transamination Bertoldi et al. (1999)100 AAS Plants and Fungi PF00282 Tyrosine/DOPA/Serine Decarboxylase Oxidative Deamination Kaminaga et al. (2006),101 Sakai et al. (2007),102 Torrens-Spence et al. (2012),103 Torrens-Spence et al. (2018),104–106 Gutensohn et al. (2011)107 and Torrens-Spence et al. (2014)108 AAS Insects PF00282 Tyrosine/DOPA Decarboxylase Oxidative Deamination Vavricka et al. (2011)109 ODC (paracatalytic) PF02784 Ornithine Decarboxylase Decarboxylation/Oxidative Deamination/Abortive Transamination Bertoldi et al. (1999),100 Sakai et al. (1997)110 and Pontrelli et al. (2018)111 CcbF PF00155 Aspartate Amino Transferase Oxidative Deamination Wang et al. (2016)112 Ind4 PF00155 Aspartate Amino Transferase Arginine Desaturation Du et al. (2016)113 MppP PF00155 Aspartate Amino Transferase Arginine Hydroxylation Han et al. (2015)114 and Han et al. (2018)115 RohP PF00155 Aspartate Amino Transferase Arginine Hydroxylation Hedges et al. (2018)116 CuaB PF00155 8-amino-7-oxononanoate synthase Decarboxylating Oxygenation Dai et al. (2020)117 PvdN PF00266 Cysteine Desulferase, CsdA Decarboxylating Oxygenation Ringel et al. (2016)118 Cap15 PF03841 Selenocysteine Synthase, SelA Decarboxylating Oxygenation Huang et al. (2018)119 *Protein Family and Predicted Function were determined from a combination of the cited literature and BLAST search predictions of each enzyme. 21 1.4.1 Understanding reactivity of PLP-dependent enzymes with O2 The chemical reasons that PLP-dependent enzymes use oxygen in reactions are not yet fully understood. Oxygen, which normally exists in the triplet state, is fairly unreactive towards organic cofactors, typically found in a singlet state, as this is a spin-forbidden process. Therefore, enzymes catalyzing such reactions must be able to overcome this challenge. Flavin-dependent enzymes are a ubiquitous group of enzymes that commonly react with oxygen and are often used as a model to understand enzymatic oxygen reactivity in the absence of a metal cofactor. In these reactions, the need for intermediate spin inversion is overcome by an initial single electron transfer from reduced flavin to the triplet state oxygen, generating superoxide and a flavin semi-quinone as a caged radical pair.120,121 This process can be understood through Marcus theory, in which electron transfers are governed by the redox potentials of the reactants and the reorganization energy,122 the latter of which is thought to be the more important contributor to flavin-oxygen reactivity in flavo-proteins.120 Active site polarity, confined oxygen binding sites and flavin stereochemistry are specific contributors to lowering the reorganization energy in flavo-proteins resulting in enhanced oxygen reactivity.120,121 Studies of cofactor-independent oxygenases, which employ carbanions, used flavo-proteins as a model to understand oxygen reactivity. Experimental observation of radical intermediates by electron paramagnetic resonance (EPR) in a cofactor-independent dioxygenase suggested that the chemistry of cofactor-independent oxygenases likely traverses through carbanionic donation of a single electron to dioxygen.123 It is conceivable that something similar to the flavo-protein mechanism of oxygen activation could be occurring within some PLP-dependent decarboxylases (Figure 1.8). Most mechanistic proposals96,124–127 describe a streamlined process in which the quinonoid intermediate donates a single electron to oxygen, forming superoxide and a semiquinone as a caged radical pair. Then, the radical pair can collapse to form a hydroperoxyl-intermediate localized at either the C4′ of PLP (Hydroperoxy I) or the a-carbon of the substrate (Hydroperoxy II). Eliminating the hydroperoxyl-intermediate forms hydrogen peroxide and an imine product, which hydrolyzes in water to release ammonia and a carbonyl-containing molecule. Flavoprotein investigations also suggest that formation of the hydroperoxyl-intermediate may vary between enzymes, which could also be the case for the PLP-dependent enzymes.120,121 22 Although it is predicted that the PLP-dependent oxygen reactivity will likely proceed through a single electron transfer, it is theoretically possible to activate a carbanionic intermediate by excitation to the triplet state. In the triplet state, the intermediate reaction with oxygen would no longer be spin forbidden. Those studying oxygen activation by cofactor-independent enzymes also discussed a similar possibility prior to obtaining experimental support for a single electron transfer process.123,128 At this time neither hypotheses can be disregarded for the PLP-dependent oxygen reactivity due to a lack of evidence. It is hoped that this discussion will inspire others to further investigate the oxygen reactivity both experimentally and computationally. NHNOH2-O3POR OOHNHNOH2-O3POR HNHNOH2-O3POR HNHNOH2-O3POR HNHNOH2-O3POR HOOHOHONHNOH2-O3POLys-EnzHROO2O2H+H2O2H2ONH3CO2H+ExternalAldimine QuinonoidRadical PairHydroperoxy IHydroperoxy IIH2OH2O2NH3InternalAldimineFigure 1.8: Proposed mechanism for paracatalytic oxidative deamination from PLP-dependent decarboxylases. 23 1.4.2 Paracatalytic reactions of PLP-dependent enzymes with O2 The term ‘paracatalytic’ describes the use of a non-physiological oxidizing reagent in an enzymatic reaction,96–98 a term that also encompasses oxidative side-reactions with O2.129 The first enzyme found to undergo a paracatalytic reaction with O2 itself was Rubisco, which produces two molecules of 3-phosphoglycerate from ribulose-1,5-bisphosphate, but will also use O2, paracatalytically, to make one molecule of 3-phosphoglycerate and one molecule of 2-phosphoglycerate.130,131 Since the discovery of Rubisco’s paracatalytic activity, several other enzymes were also found to have paracatalytic reactions with oxygen, including glutamate decarboxylase, which is the first PLP-dependent enzyme discovered to react with oxygen.95 Until recently, knowledge of PLP-dependent reactions with oxygen were limited to paracatalytic activities. Paracatalytic reactions with oxygen, including and extending beyond the PLP-dependent examples, have been reviewed previously.96 There are three types of known paracatalytic PLP-dependent enzymes that react with oxygen: 3,4-dihydroxyphenylalanine (DOPA) decarboxylase, glutamate decarboxylase (GAD) and ornithine decarboxylase (ODC). DOPA decarboxylase (DDC) catalyzes the conversion of aromatic L-amino acids L-DOPA and L-hydroxytryptophan (L-HTP) into the corresponding amines via decarboxylation (Figure 1.9A and Figure 1.10A). Additionally, DDC has two additional reactions: abortive transamination and oxidative deamination (Figure 1.9A and B).99 Abortive transamination proceeds through protonation of the quinonoid at the C4′ position, giving the ketimine, which is subject to hydrolysis, releasing the corresponding aldehydes or ketones and pyridoxamine 5′-phosphate (PMP) (Figure 1.9B). Oxidative deamination by DDC also gives the corresponding aldehydes or ketones but through paracatalytic reactions with O2, regenerating PLP instead of forming PMP (Figure 1.9C).132 Similarly, GAD from E. coli and ODC from Hafnia alvei and Lactobacillus 30a also exhibit abortive transamination and oxidative deamination in addition to their typical decarboxylation reactions (Figure 1.10B and C).95,100,110 Interestingly, it has been shown that the reactivity of some of these decarboxylases can be modulated to favour oxidative deamination by mutating certain residues. The pig kidney DDC Y332F variant exclusively catalyzes oxidative deamination under aerobic conditions and abortive deamination under anaerobic conditions, with abolished decarboxylation activity.126 The predicted function of Tyr332 is as a proton donor for the substrate a-carbon following decarboxylation. Such proton donation which would no longer be possible in the Y332F variant, 24 thus leading to oxidative deamination.126 This claim is supported by a crystal structure for human GAD67 which showed that Tyr434 (equivalent to DDC Tyr332) is positioned to protonate the a-carbon with the help of a nearby histidine in Chain A, supporting that the tyrosine residue could be responsible for modulating oxygen reactivity through a-carbon protonation (Figure 1.11A).133 However, Chain B shows Tyr434 interacting with the backbone amine of Tyr292, while His291 is closer to the a-carbon of the substrate, suggesting that His291 could act as the proton donor (Figure 1.11B).133 More recently, ODC from E. coli, SpeC, was found to accumulate mutations, G655A and G655S, that converted the decarboxylase into a dedicated oxidative transaminase in response to an ʟ-alanine auxotrophic mutation, ΔpanD.111 Wild-type SpeC also demonstrated some oxidative deamination activity, additional to the expected decarboxylase activity, but an explanation for why these mutations might cause the change in reactivity has not yet been proposed. 25 NHNHOH2-O3PORC⍺C4’NHNOOROH2-O3PONH3LysExternalAldimineHNHNOORNH3OH2-O3POLysInternalAldimineC⍺C4’NHNHOH2-O3PORNHNHOH2-O3PORNHNHOH2-O3PORHNHNRNH2OH2-O3POLysInternalAldimineQuinonoidHHNHNH2ROOH2-O3POPMPHROHNHNOH2-O3POLysInternalAldimineOHOCO2H+O2H+H2OH2OH2O2Decarboxylation Oxidative DeaminationAbortive TransaminationNH3H+H+HHH HABCFigure 1.9: Mechanisms of decarboxylation, abortive transamination and oxidative deamination in the paracatalytic decarboxylases. All mechanisms traverse through a decarboxylated quinonoid intermediate and are differentiated by the site of reprotonation or by reaction with oxygen. 26 CO2DecarboxylationOxidativeDeaminationAbortiveDeaminationPLPCO2PLPCO2PLPNH3H2O2CO2PMPOH2NNH3Oʟ-ornithineH3NNH3putrescine1-pyrrolineNN1-pyrrolineBNH3OOO OO2DecarboxylationOxidativeDeaminationAbortiveDeaminationʟ-glutamatePLPNH3OO훾-aminobutyric acidCO2PLPOOOsuccininc semialdehydeCO2PLPNH3H2O2OOOsuccininc semialdehydeCO2PMPAHOHO NH3OOʟ-DOPAHOHONH3DopamineCO2O2PLPPLPHOHOO3,4-dihydroxyphenylacetaldehydeCO2PLPHOHOOCO2PMPDecarboxylationOxidativeDeaminationAbortiveDeaminationNH3H2O23,4-dihydroxyphenylacetaldehydeFigure 1.10: Overall schemes for the three types of PLP-dependent decarboxylases with paracatalytic reactivity. (A) DOPA decarboxylase (DDC). (B) Glutamate decarboxylase (GAD). (C) Ornithine decarboxylase (ODC). Each scheme shows the substrate and products for decarboxylation, oxidative deamination, and abortive deamination. Predicted, but unconfirmed species are indicated by a dashed box. 27 1.4.3 Oxidative deamination 1.4.3.1 Plant amino acid aldehyde synthases The first PLP-dependent enzyme discovered to have a dedicated oxygen reactivity was the phenylacetaldehyde synthase (PAAS) from Petunia hybrida cv. Mitchell.101 Phenylacetaldehyde (PHA) is an important scent compound, giving fragrance to flowers, fruits and some processed foods, such as bread and cheese.134,135 Kaminaga et al. found that the petunia PAAS produces stoichiometric amounts of carbon dioxide, ammonia, hydrogen peroxide and PHA, paired with oxygen consumption (Figure 1.12A) and that decarboxylation precedes deamination in the mechanism.101 Similarly, the rose PAAS produces PHA, ammonia and hydrogen peroxide in nearly stoichiometric amounts under aerobic conditions.102 Since the discovery of the rose and petunia PAASs, more examples of amino acid aldehyde synthases (AASs) with activity on various substrates have been discovered from plants, including from parsley (Petroselinum crispum),103 Rhodiola rose,104 Arabidopsis thaliana,107 Cicer arietinum (chickpeas),108 Medicago truncatula108 and Eucalyptus grandis106 (Table 1.1). The first known fungal AAS example, 3,4-dihydroxyphenylacetaldehyde (DHPAA) synthase, has also been discovered from Psilocybe cubensis.105 A BFigure 1.11: X-ray crystal structure of human GAD67 (PDB code: 2OKJ). Each active site, the A-chain (A) or the B-chain (B), from the dimer is shown. The A-chain is shown in light green while the B-chain is shown in dark green. A trapped intermediate (PLP-GABA) from the B-chain active site is coloured magenta. PLP-GABA from Chain B was superposed into Chain A for easier comparison of the structures. Distances between atoms are shown by dashed lines and are given in Å. Water molecules are shown as red spheres. 28 A study comparing Arabidopsis thaliana and parsley AASs with similar amino acid decarboxylases (AADCs) in Catharanthus roseus and Papaver somniferum, which do not catalyze oxidative deaminations, aimed to understand the distinguishing features between the two groups of enzymes.136 An active site tyrosine residue is conserved in the decarboxylases, while the corresponding residue is a phenylalanine or valine in the AASs (Figure 1.13A). Mutating the active site phenylalanine to a tyrosine in the AAS resulted in AADC activity, and vice versa, demonstrating the importance of this residue on distinguishing the reactivity. This result is consistent with work on the paracatalytic DDC Y344F variant.126 The results also offered a new hypothesis for the role of the tyrosine, suggesting that it might stabilize ionization of a neighbouring, conserved histidine residue while the histidine itself protonates the a-carbon.136 The AAS from Eucalyptus grandis contains a histidine to asparagine substitution, which supports the importance of this conserved histidine, but molecular dynamics simulations in this study suggested the tyrosine as the proton donor.106 OOH3N OHO HOHO HOAASO2 H2O2NH3CO2OONH3AASHOHOHOHOHONH3HOHOAADCO2H2O2NH3CO2CO2B Flexible, colourlesscuticleRigid, pigmented cuticleOONH3PAAS HOPAROHO2 H2O2NH3CO2NADPH NADP+AFigure 1.12: Schemes for physiological roles of major AAS enzymes are shown. (A) Plant PAAS involvement in producing 2-phenylethanol from L-phenylalanine. (B) Insect AAS involvement proposed in the formation and flexible, colourless cuticle from L-DOPA. The physiological connection between effects of a-methylDOPA feeding and observed AAS activity is also described. 29 1.4.3.2 Insect amino acid aldehyde synthases Insects have several annotated AADCs in their genomes, however one enzyme, DHPAA synthase (DHPAAS), catalyzes an oxidative deamination of ʟ-DOPA, as demonstrated in Aedes aegypti, Drosophila melanogaster, Anopheles gambiae, and Culex quinquefasciatus.109 The product of this enzyme, DHPAA, is hypothesized to be important in uncoloured, flexible cuticle AaAASDmAAS1DmAAS2CqAASAaAADCDmAADCAgAADCS D Q S N S A V ES D Q S N S C I ES D Q S N S C I ES D Q S N S A V ES N Q S H S S V ES D Q A H S S V ES N Q S H S S V E191188188148188188226199196196156196196234.....................331328328288328327365339336336296336335373V D R I Y L Q H KV D R I Y L K H KV D R I Y L K H KV D R I Y L Q H KV D P L Y L K H DV D P L Y L K H DV D P L Y L K H DBPhAASRhAASAtAASPcAASMtAASCaAASPsAADCTfAADCAtAADCCrAADCOsAADCD Q T H F S FD Q T H S T LD Q T H S A LD Q T H S A LQ D S H Y S IQ D S H Y S IN Q T H C A LD Q T H C A LD Q T H S S FD Q T H T M FD Q T H S T F199200190198186159202199242200211205206196204192165208205248206217T N P E V L R N DT N P E F L R N KT N P E F L K N KT Y P E F L K N NR D V D I I A S RR D V E I I A S RT S A E Y L K N KT N P E Y L R N KT N P E Y L E F KT N P E Y L K N KT N P E Y L K N H343344334342325298346343386344355351352342350333306354351394352363.................................AFigure 1.13: Partial sequence alignments comparisons of AASs and AADCs in (A) plants and (B) insects. Alignments were made with Clustal Omega in UGENE.175 Arrows indicate the proposed catalytic residues responsible for protonating the a-carbon in the decarboxylases in both the plants and insects. A filled in arrow indicates the key residue responsible for modulating oxidative deaminase activity. (A) Plant sequences [PhAAS: Petunia hybrida cv. Mitchell (ABB72475.1), RhAAS: Rosa hybrid cultivar (ABB04522.1), AtAAS: Arabidopsis thaliana (NP_849999.1), PcAAS: Petroselinum crispum (Q06086.1), MtAAS: Medicago truncatula (XP_003592128.2), CaAAS: Cicer arietinum (XP_004496485.1), PsAADC: Papaver somniferum (AAC61842.1), TfAADC: Thalictrum flavum subsp. Glaucum (AAG60665.1), AtAAS: Arabidopsis thaliana (NP_001078461.1), CrAADC: Catharanthus roseus (P17770.1), OsAADC: Oryza sativa (BAG91223.1)] are aligned to show catalytic residues that distinguish oxidative from non-oxidative reactivity. (B) Insects sequences [AaAAS: Aedes aegypti (EAT37247.1), DmAAS1: Drosophila melanogaster (NP_476592.1), DmAAS2: Drosophila melanogaster (NP_724162.1), AgAAS: Anopheles gambiae (XP_319838.3), CgAAS: Culex quinquefasciatus (EDS39158.1), AaAADC: Aedes aegypti (XP_001648264.1), DmAADC: Drosophila melanogaster (NP_724164.1), AgAADC: Anopheles gambiae (AAC16249.1)] are aligned to show catalytic residues that distinguish oxidative from non-oxidative reactivity. Figure was adapted from Hoffarth et al. (2020). 30 formation in insects, perhaps through aiding crosslinking of cuticular proteins (Figure 1.12B).109,137 The gene responsible for producing DHPAAS in Drosophila had been previously designated the a-methyldopa hypersensitive (amd) gene, based on observed sensitivity to a-methylDOPA (AMD) when the gene was mutated.138–141 The mutants have defects in cuticle development, but the function of the amd gene was unclear at the time. The insect DHPAAS was able to convert AMD to 3,4-dihydroxyphenylacetone, demonstrating a link between the observed phenotype and the enzymatic function (Figure 1.12B).109 Liang et al. produced homology models to compare an AADC with an AAS from Drosophila and observed that active site His192 was associated with decarboxylation, whereas Asn192 in the corresponding position resulted in oxidative deamination (Figure 1.13B).142 The importance of this residue was confirmed by mutating the His in AADC to Asn, and the Asn in AAS to His, resulting in loss of oxidative deamination in the Asn to His mutant and gain of oxidative deamination in the His to Asn mutant. The authors propose that the His192 is able to protonate the a-carbon to facilitate decarboxylation, while the asparagine residue cannot. Another study aimed at optimizing the decarboxylation and oxidative deamination reactivity of insect AAS for improved biosynthesis of benzylisoquinonline alkaloids supported the key role of Asn192, but also found that Phe79 and Tyr80 are important for modulating this activity.143 Although, with the exception of Eucalyptus grandis,106 the key residue implicated with distinguishing AADCs from AASs in insects is not the same as in plants, but these residues are not unrelated because the catalytic His192 residue in the insect AADC corresponds to the same active site histidine residue described in Section 1.4.3.1 for plants (Figure 1.13). Therefore, it seems that each species took a different evolutionary pathway by mutating a different residue to converge on the same outcome. Any disruption to the catalytic histidine or tyrosine residues seems to result in oxidative deamination, exclusively, or bifunctionally with decarboxylation.106 1.4.3.3 Lincosamide biosynthetic enzyme CcbF Lincosamide antibiotics inhibit protein synthesis in gram-negative cocci and in gram-positive cocci and bacilli.144 Structurally, they are characterized by an eight-carbon amino sugar with a sulfur appendage linked to an amino acid moiety by an amide bond. Lincomycin and celesticetin are lincosamide antibiotics from Streptomyces lincolnensis and Streptomyces caelestis, respectively, that differ in regard to their S-substitution: where lincomycin contains a 31 methylsulfhydyl moiety, and celestietin contains a salicylate moiety attached to sulfur via a 2-carbon chain (Figure 1.14). The biosynthetic gene clusters differ by a pair of homologous PLP-dependent enzymes, LmbF and CcbF (Figure 1.14A).112,145 The lincomycin biosynthetic enzyme, LmbF, is responsible for C-S bond cleavage, removing most of the S-cysteine residue through a PLP-dependent β-elimination reaction, while the celesticetin biosynthesis enzyme CcbF was predicted to process the S-cysteine residue, through decarboxylation-dependent transamination.145 In an effort to confirm the proposed decarboxylation-dependent transamination reaction, Ushimaru and colleagues further investigated CcbF, and confirmed formation of the aldehyde product, but could not detect formation of PMP or use of a keto-acid co-substrate to regenerate PLP, indicating that catalysis proceeds through another mechanism.146 Wang and colleagues found that oxygen is essential for CcbF enzymatic activity, and that hydrogen peroxide, ammonia and carbon dioxide are generated along with the product.112 These findings led the authors to propose a mechanism in which decarboxylation of the external aldimine intermediate results in a quinonoid intermediate that is susceptible to react with oxygen, producing hydrogen peroxide followed by imine hydrolysis of the product (Figure 1.14B). 32 1.4.4 Decarboxylating oxygenation 1.4.4.1 PvdN Pyoverdines are molecules composed of a 2,3-diamino-6,7-dihydroxyquinoline linked to a peptide moiety which play a role in virulence and toxicity in pathogenic Pseudomonas species.147–149 Of the five enzymes involved in the biosynthetic pathway to pyoverdine (PvdM, PvdN, PvdO, PvdP, and PvdQ), only the roles of PvdP and PvdQ were known until recently.150–152 Ringel and colleagues investigated the catalytic role of PvdN, demonstrating that it converts a glutamic acid moiety at the 3-amino group of the pyoverdine chromophore to a succinamide NHOH2-O3PONNHOH2-O3POO2OOH3NS OOSNHSCO2H2O2NH2SH2OOSNHOH2-O3PONLysNH3NHOH2-O3PONLysNONHOSOHOHOHOHLincomycinCelesticetinRNONHOSOHOHOHOHNH2HOOLmbFCcbFNONHOHSOHOHOHOHLmbGCysteine S-conjugated IntermediateNONHOSOHOHOHOHOCcb1-5Linconomycin A: R = n-PrCelestecetin: R = HNONHOSOHOHOHOOOOHO2H2O2NH3CO2ABFigure 1.14: Biosynthesis of lincomycins and celesticetins. (A) Branching point catalyzed by LmbF and CcbF in lincomycin and celesticetin biosynthesis, respectively. (B) Proposed mechanism for CcbF. 33 group via a ΔpvdN mutation and pvdN complementation (Figure 1.15A and Figure 1.15B).118 The authors postulated that the conversion to succinamide could occur via an O2-, PLP-dependent oxygenation-decarboxylation cascade, in which PvdN forms a Schiff base between the PLP-cofactor and the a-amino nitrogen of the glutamic acid moiety (Figure 1.15C). Decarboxylation forms a quinonoid intermediate, which reacts with a molecule of oxygen, forming a peroxy-adduct at the glutamic acid a-carbon. As the peroxy-adduct intermediate degrades through breakage of the oxygen-oxygen bond, the amide group of the succinamide moiety observed in pyoverdine is formed. The structure of PvdN supports this proposal through the existence of two tunnels connecting the enzyme’s surface to active site, one of which is large enough for the substrate, while the other is narrower to accommodate the exchange of CO2 and O2.118,153 Future work is needed to assess the validity of this proposal through in vitro studies, particularly to demonstrate the enzyme’s reliance on oxygen. 34 1.4.4.2 Cap15 Capuramycins are a class of nucleoside antibiotics originally isolated from Streptomyces griseus.154 Structurally, this class of compounds is characterized by a uridine 5′-carboxamide core appended to an unsaturated hexuronic acid through a glyosidic linkage (Figure 1.16A). The biosynthetic gene clusters for capuramycin natural products have been identified.155,156 The final step in the biosynthesis of the uridine 5′-carboxoamide (CarU) core involves the conversion of epimerized 5′-glycyluridine (GlyU) catalyzed by Cap15 (Figure 1.16B). In vitro assays showed OONH3NHOH2-O3PONLys-PvdNNHOH2-O3PONNHOH2-O3PONOONHOH2-O3PONO2OHONHOH2-O3PONOHOBHBHH2NO H2OCO2NH2OOOOOONH2ΔpvdNONH2ΔpvdN/pvdN complementWTON OHO NHHNOOHHNNHOONOHOHNONHOH2NHNOHNHOOHN NNHOHOHROPVDA506CA BFigure 1.15: Pyoverdine and proposed mechanism of PvdN. (A) Structure of pyoverdine, PVDA506 with the location of the PvdN-modified substituent indicated by an R-group. (B) Biosynthesis of succinamide and a-ketoglutarate substituents from glutamic acid in wild-type P. fluorescens strain A506, ΔpvdN mutant strain, and ΔpvdN mutant strain complemented with pvdN. (C) Proposed reaction mechanism for PvdN. 35 that Cap15 is oxygen-dependent because the reaction did not proceed in anaerobic conditions.119 An isotopic labelling experiment using either H3P18O4, H218O or 18O2 established Cap15 as a monooxygenase, since isotopic enrichment was only observed in reactions with 18O2. CO2 was also detected in the reaction, which further classifies Cap15 as a monooxygenase-decarboxylase. The authors proposed a mechanism for Cap15 based on ultraviolet-visible spectroscopy (Figure 1.17), in which removal of the a-carbon forms a quinonoid intermediate capable or reacting with oxygen. Decarboxylation then occurs, eliminating the distal hydroperoxide oxygen in the form of water, and yielding a CarU-PLP aldimine that then hydrolyzes to release CarU. O N NHOOOHHOOPOHO OCapA O N NHOOOHHOOH HCapH O N NHOOOHHOHHOOHOH2N?O N NHOOOHHOHHOOHOH2NO N NHOOOHHOHHOH2N Oα-KGO2 H2PO4-CO2Succinate AcetaldehydeCO2CarUCap15ʟ-ThrH2PO4-O2NNHOOOOMeOOH2N OOOHOHONHHNOA-503083 ANH2ONNHOOOOHMeOOH2N OOOHOHONHHNOA-500359 AA-102395NNHOOOOHMeOOH2N OOOHOHONHOHOHNHONHOHOOABFigure 1.16: Structures and biosynthesis of capuramycin-type molecules. (A) Example structures of capuramycin-type metabolites with shared CarU core shown in blue. (B) Biosynthesis of CarU core. 36 1.4.4.3 CuaB Indolizidine alkaloids are a bicyclic-containing natural product group of molecules with antibacterial, antiinflamitory and antitumor bioactivities.157–159 Curvulamine is an antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10, a fungus associated with the white croaker.160 During analysis of curvulamine biosynthesis, a novel bifunctional PLP-dependent enzyme, CuaB, was found to be essential to the formation of the bicyclic indolozidine skeleton.117 First, CuaB catalyzed a Claisen condensation reaction between an acyl carrier protein-bound tetraketide and alanine, similar to the previously known 8-amino-7-oxononanoate synthase (AONS). Then, CuaB catalyzes an oxygen-dependent hydroxylation and cyclization, which was confirmed by isotopic labelling experiments using either H218O or 18O2, the latter of which showed isotopic enhancement in the products. The oxygen-derived hydroxylation appears at the a-carbon of the alanine residue of the Claisen condensation product, suggesting that a hydroperoxyl intermediate forms at this location. Cap15 OHLysN2-O3PONH(5’S,6’R)-GlyU BHOOHNNHOHHOHO OOOOHN2-O3PONHNH2-O3PONOHR OOHOO2O2HO OOROHN2-O3PONHHBOHOHO OOROHN2-O3POH+NHCO2HOHHOROOHN2-O3PONHPLPCarU391 nm 416 nm503 nmFigure 1.17: Proposed mechanism of Cap15, including observed UV spectra assigned to predicted intermediates. 37 1.4.5 Arginine oxidation 1.4.5.1 MppP Enduracididine is an arginine-derived, non-proteinogenic, guanidine-containing, heterocyclic amino acid and is a biosynthetic precursor to teixobactin,161 enduracidin,162 and mannopeptimycin,163 which are antibiotics against a variety of bacteria (Figure 1.18). Genes for making this non-canonical amino acid were proposed during the discovery of the enduracidin gene cluster164 through comparison with the mannopeptimycin gene cluster;165 however, the function of these genes was established by Silvaggi through in vitro and crystallographic characterization of enzymes in the pathway114,115,166 (Figure 1.18). First, a PLP-dependent oxidase, MppP/EndP, converts L-arginine into 4-hydroxy-2-ketoarginine,114,115 which is dehydrated and cyclized by MppR/EndR,166 and proposed to be aminated by MppQ/EndQ, another PLP-dependent enzyme. Enduracididine is sometimes further hydroxylated at the a-position by MppO, as is seen in mannopeptimycin,165,167 before being incorporated into a natural product by a non-ribosomal peptide synthetase. Further studies of MppP114,115 found that it was an oxygen- and PLP-dependent arginine oxidase that catalyzes a two- and four-electron oxidation to produce 2-ketoarginine and (S)-4-hydroxy-2-ketoarginine, respectively, with the hydroxyl group on the (S)-4-hydroxy-2-ketoarginine product coming from water (Figure 1.19). The stereochemistry of this product was established using an X-ray crystal structure of MppP soaked with a mixture of reaction products, allowing the 4-hydroxy-2-ketoarginine to bind to the active site.115 The enzyme uses one or two oxygen molecules, which are stoichiometrically converted to hydrogen peroxide.115 38 NHH2N NH OOONHH2N NH NH3OOMppP2 O2 OHHN NHNHO OOHN NHNHH3NOOMppR MppQH HH2O2 H2O2NH3HNOHNHNONHONH2OHNONHO HNOHN OOONHO NHOHNOHN NHHNOHOHTeixobactinNHHOOO ONHHO2CHNOHNONHOOHHNOHNHN NHHNONHO HNClOHClONHOHO HNOHOHNHNNHNHONHNHH2NO O OHNHOHNOHONHOHOHNOHNH2OEnduracidin AONHNHOOHNHNHNOHOOHOHOHHNOHNHHNNHOHNOOHNOHO NHOO OOOHHOOHOHOHHOOHMannopeptimycin ⍺mppPmppO mppQ mppR1 kbendP endQ endRStreptomyces hygroscopicusStreptomyces fungicidicusFigure 1.18: The enduracididine biosynthetic pathway and gene cluster. MppP, an O2- and PLP-dependent enzyme, converts ʟ-arginine to (S)-4-hydroxy-2-ketoarginine during enduracididine biosynthesis. Enduracididine-containing natural products are also shown with the enduracididine moiety highlighted in blue. Figure adapted from Hoffarth et al. (2020). 39 1.4.5.2 RohP Azomycin, or 2-nitroimidazole, inspired a suite of synthetic nitroimidazole drugs for antimicrobial use.168 Biosynthetic studies of this molecule revealed that 2-aminoimidazole is a precursor to azomycin.169–171 In 1977, Eguchi demonstrated that 4-hydroxy-2-ketoarginine was a key precursor to 2-aminoimidazole by feeding radiolabeled arginine to crude enzyme fractions of Streptomyces eurocidicus.172 The authors suggested that an unknown enzyme could catalyze a retro-aldol type reaction on 4-hydroxy-2-ketoarginine to produce pyruvate and NHOH2-O3PONOONHH2NNH2 HNHOH2-O3PONOONHH2NNH2LysNH2Quinonoid I NHOH2-O3PONOONHH2NNH2BNHOH2-O3PONOONHH2NNH2BH HOONHH2NNH2ONHOH2-O3PONOONHH2NNH2Quinonoid IIOONHH2NNH2NHOONHH2NNH2OH Oreleasefrom cofactorH2OO2H2O2O2NHOH2-O3PONOONHH2NNH2releasefrom cofactorOONHH2NNH2OH2OH2O2C⍺C⍺C4′C⍺NHOH2-O3PONOONHH2NNH2OH2-ketoarginine4,5-dehydro-2-ketoarginine4,5-dehydro-2-iminoarginine(S)-4-hydroxy-2-ketoarginineC4′C4′NH3NH3O2H2O2H2Oreleasefrom cofactorH2ONH3Desaturation(Ind4)Hydroxylation(MppP, RohP)Figure 1.19: Proposed mechanism for known arginine oxidase products. Observed products are indicated with a box. The pathway leading to 2-ketoarginine is indicated with a blue reaction arrow, while the pathway leading to the second oxidation step through quinonoid II is indicated with red reaction arrows. The branching point between desaturation and hydroxylation is shown with green and purple reaction arrows, respectively. 40 guanidinoacetaldehyde, which would lead to a guanidine cyclization and formation of 2-aminoimidazole. They also showed that the production of 4-hydroxy-2-ketoarginine was dependent on oxygen and PLP. This proposal was recently linked to a group of biosynthetic enzymes by the Ryan group173. The biosynthetic pathway begins with arginine being converted to 4-hydroxy-2-ketoarginine by RohP.116 Then RohR catalyzes a retro-aldol reaction on 4-hydroxy-2-ketoarginine to produce pyruvate and guanidinoacetaldehyde, which is cyclized by RohQ to 2-aminoimidazole. RohS then oxidizes the 2′-amino group to form azomycin (Figure 1.20). RohP was characterized as an oxygen- and PLP-dependent arginine oxidase through in vitro characterization, which revealed that oxygen is consumed while 2-ketoarginine, 4-hydroxy-2-ketoarginine and H2O2 are produced, similar to what was observed in MppP.116 H2O2 was produced stoichiometrically to oxygen consumption. Additionally, reactions in 50% H218O demonstrated that the hydroxyl group is derived from water rather than hydrogen peroxide (Figure 1.19). Crystal structures of this enzyme have been solved for the holoenzyme, two intermediates (quinonoid I and quinonoid II), and the product-bound enzyme. The configuration of the hydroxyl group was established as (S)-4-hydroxy-2-ketoarginine using a product-bound crystal structure. This enzyme plays a key role in the biosynthesis of the azomycin heterocycle because it allows for a retro-aldol reaction to occur in the next step of biosynthesis, after which guanidinoacetaldehyde is capable of cyclizing into 2-aminoimidazole either spontaneously, or by enzymatic catalysis from RohQ. H2N NHNH2NH3OOH2N NHNH2OOOOHRohP RohRH2N NHNH2OHOOORohQH2ONHNNH2NHNNO2RohS2 O2 2 H2O2H2OO2NH3rohTrohP rohQ rohR rohS0.5 kbFigure 1.20: The azomycin gene cluster and biosynthetic pathway. RohP, an O2- and PLP-dependent enzyme, converts ʟ-arginine to (S)-4-hydroxy-2-ketoarginine during azomycin biosynthesis. Figure adapted from Hoffarth et al. (2020). 41 1.4.5.3 Ind4 Indolmycin, described earlier in Section 1.2, is an antibiotic that was originally discovered in 196028 and inhibits prokaryotic TrpRS.29 It consists of an indole moiety linked to an oxazolinone heterocycle, which is formed by the ATP-dependent condensation of indolmycenic acid and D-4,5-dehydroarginine (Figure 1.5). The production of D-4,5-dehydroarginine is dependent on Ind4, an oxygen- and PLP-dependent oxidase, and Ind5, a stereospecific nicotinamide adenine dinucleotide phosphate (NADPH)-dependent dehydrogenase, respectively113,174. The double bond formed adjacent to the guanidine group is essential to the formation of the oxazolinone ring, as arginine cannot be used in this transformation, and the adjacent double bond probably plays a role in increasing the electron density toward the guanidine group, allowing one of the nitrogens to act as a nucleophile towards the adenylated carboxylic acid. Ind4 utilizes one or two molecules of oxygen to catalyze a two- or four-electron oxidation of arginine, producing 2-ketoarginine and 4,5-dehydro-2-iminoarginine, respectively (Figure 1.19). Stoichiometric hydrogen peroxide is produced for every molecule of oxygen consumed. The reaction does not proceed in the absence of oxygen and does not require the addition of external co-factor or a-ketoglutarate to regenerate PLP from PMP. Through spectroscopic stopped-flow analysis, a three-step model was proposed to best fit the observed kinetic data under aerobic conditions. In this model, catalysis involves formation of a quinonoid intermediate, stable in the absence of oxygen; formation of a second more oxidized quinonoid intermediate; and conversion of the more oxidized quinonoid into a highly conjugated product-PLP complex. Stopped flow analysis under anaerobic conditions demonstrated accumulation of the first quinonoid intermediate, suggesting that the quinonoid species is reactive with oxygen. In the biosynthetic pathway, the Ind56 complex then stereospecifically reduces the imino acid products to D-configured amino acids, which are not substrates of Ind4. In addition to the indolmycin biosynthetic gene cluster, a number of other non-indolmycin gene clusters contain paired ind45 gene homologs,113 suggesting that the functions of Ind4- and Ind5-like enzymes are also linked in other biosynthetic pathways. 42 1.5 Aims of study Studies in natural product biosynthesis show the importance of exploring natural systems in order to discover new and useful tools that can be added to the biocatalytic repertoire. Previous discovery of the indolmycin biosynthetic machinery reveals that it may be a rich source of biocatalytic potential in two ways: expansion of known reactions catalyzed by enzymes and the ability to manipulate production of indolmycin. This work focuses on using the indolmycin biosynthetic pathway to expand the knowledge of newly discovered enzymatic reactions and to build new indolmycin analogs, which will demonstrate the biocatalytic potential of this pathway and the importance of studying natural product biosynthesis. In Chapters 2 and 3, it was hypothesized that structural, biochemical and phylogenetic studies of the arginine desaturases would facilitate a better understanding of the arginine oxidases and their mechanisms. There are two elusive aspects of the mechanism that this study aims to solve: the mechanism of oxygen usage and the differentiation of hydroxylation and desaturation. X-ray crystallography will be used to obtain the first crystal structures of an arginine desaturase and mutagenesis based on these crystal structures will be used to explore the catalytic residues of the desaturases. Additionally, the sequence similarity and phylogenetic relationship between the desaturases and hydroxylases will be analyzed to better understand the evolutionary history of these enzymes. Several new homologs of Ind4 with desaturase activity will be identified and their products will be characterized in further detail. Finally, a mechanism is proposed that unifies the experimental and calculated data for both the hydroxylases and desaturases. This information will help guide potential efforts to discover and engineer arginine oxidases and other oxygen- and PLP-dependent enzymes. Advances in oxygen- and PLP-dependent enzyme catalysis will expand the biocatalytic repertoire and aid the development of more PLP-dependent enzymes for use as biocatalysts. In Chapter 4, it was hypothesized that heterologous expression of the indolmycin biosynthetic genes in E. coli would provide a useful method of making indolmycin. Additionally, advances in the biocatalytic use of TrpS to make various tryptophan derivatives from substituted indoles and L-serine suggest that coupling this enzyme with the indolmycin biosynthetic genes, in vivo, may be a simple way to diversify indolmycin analogs. Therefore, heterologous expression of indolmycin genes, paired with a promiscuous TrpS, will be explored here for use in the production of diverse indolmycin analogs. The production of more diverse indolmycin 43 analogs may benefit the fight against antimicrobial resistance by providing more molecules that can be tested for desirable qualities. Additionally, use of this heterologous system, or parts of it, will demonstrate the importance of understanding how biosynthetic enzymes can be used effectively as biocatalysts to make new molecules that may be challenging for solely synthetic chemical methods. 44 Chapter 2: Crystallization and mutagenesis of arginine oxidases2 2.1 Introduction As described in Chapter 1, the arginine oxidases are a group of oxygen- and PLP-dependent enzymes that catalyze two types of reactions: arginine hydroxylation and arginine desaturation. The hydroxylases, RohP and MppP, use two molecules of molecular oxygen to produce one molecule of (S)-4-hydroxy-2-ketoarginine (3) and two molecules of hydrogen peroxide from L-arginine (1) or one molecule of molecular oxygen to produce one molecule of 2-ketoarginine (4) and one molecule of hydrogen peroxide from 1 (Figure 2.1).114–116 Ind4, a desaturase, uses two molecules of molecular oxygen to produce one molecule of 4,5-dehydro-2-iminoarginine (2), converting to 4,5-dehydro-2-ketoarginine (5) under longer reaction times, and two molecules of hydrogen peroxide from 1. Ind4 also consumes one molecule of molecular oxygen to produce one molecule of 4 and one molecule of hydrogen peroxide from 1 (Figure 2.1). Despite catalyzing different reactions, the hydroxylase and desaturase mechanisms are expected to be related, based on their similar usage of a PLP cofactor, stoichiometric conversion of oxygen to hydrogen peroxide and production of 4 when only one molecule of oxygen is used. Additionally, the hydroxylases share between 33%-42% sequence identity with Ind4, which will be explored with further detail in Chapter 3. The nature of the (potentially) shared mechanism within the arginine oxidases has not been adequately studied, primarily due to a lack of structural information from the desaturases. Crystal structures have been previously solved for both SwMppP and RohP, both of which showed that N-terminal ordering occurred as a result of substrate binding and was important for catalysis.114–116 Additionally, it was shown that an active site His residue was important in the production of 3.116 There were two methods used to trap intermediates of catalysis in the arginine hydroxylases. In SwMppP, an external aldimine intermediate was formed when 1 was soaked into the crystal, but a product-bound structure was obtained by soaking the crystal with reaction products.115 By contrast in RohP, intermediate-bound and product-bound structures were obtained by forming the intermediate and product ligands in crystallo by soaking 1 into the crystals and freezing each at different time points.116 Each time point and trapped intermediate corresponded to a different point in catalysis and 2 Adapted from a manuscript that is in revision for publication. 45 displayed a different colour and ultraviolet-visible (UV-Vis) signal. No oxygen atoms from molecular oxygen are incorporated into the final products of the arginine oxidase products and crystallographic studies of the arginine hydroxylases did not provide other evidence of peroxy-intermediate formation during the reaction, meaning the mechanism of oxygen utilization also remains elusive. In this chapter, the first crystal structure of an arginine desaturase is described and compared with known structures of arginine hydroxylases. This structural comparison, along with sequence alignment information, was then used to generate several desaturase variants to better understand the similarities and differences between the two types of enzymes. This new information helped to identify some of the catalytic residues of the desaturases. When compared to the hydroxylases, this new structural information helps gain a deeper understanding of the mechanism and provides insight to these unique enzymes, which is invaluable in efforts to further expand the known biocatalytic repertoire. NH2H2N NH NH3OONH2H2N NH NHOONH2H2N NH NHOONH2H2N NH NHOOOHNH2H2N NH OOONH2H2N NH OOONH2H2N NH OOOOHNH2H2N NH NH3OO+ 2 O2Ind5+ H2OInd4 Plu4+ O2or+ 2 O2+ H2OMppPoror RohPorRohPInd4Plu4or+ H2O+ H2O- NH3- NH3- NH31[M+H]+ 175 m/z5[M+H]+ 172 m/z24[M+H]+ 174 m/z3[M+H]+ 190 m/z[M+H]+ 171 m/zMppP- 2 H2O2- 2 H2O2- H2O2Figure 2.1: Scheme of O2- and PLP-dependent arginine oxidase reactivities. [M+H]+ signals are given below observed products. The Ind4-coupled reaction with Ind5 from the indolmycin biosynthetic pathway is also shown. 46 2.2 Materials and methods 2.2.1 General methods, materials and strains Reagents were purchased from Sigma-Aldrich, Hampton Research, Thermo Fisher Scientific Canada, New England BioLabs (NEB), Bio-Rad, Bio Basic Inc., Gold Biotechnology, and VWR International. Columns and resins for protein purification were obtained from GE Healthcare Life Sciences. Primers were purchased from Integrated DNA Technologies (IDT). Sequencing was done through the NAPS Unit in the Sequencing and Bioinformatics Consortium at the University of British Columbia or in the DNA Sequencing Core Facility at the Center for Molecular Medicine and Therapeutics at the University of British Columbia. The strains and plasmids used in this study are listed in Table 2.1. Table 2.1: Bacterial strains and plasmids used in this study. Strain/Vector Description Source Strain E. coli DH5α General cloning host Laboratory stock E. coli BL21 (DE3) Host for protein expression Laboratory stock Plasmid pET22b Vector for protein expression in E. coli Laboratory stock pET28a Vector for protein expression in E. coli Laboratory stock pET22b-ind4 Vector for Ind4 expression, cloning sites NdeI/XhoI Du et al.48 pET22b-plu4 Vector for Plu4 expression, cloning sites NdeI/XhoI This study pET22b-plu4H27A Vector for Plu4 variant H27A expression This study pET22b-plu4D225A Vector for Plu4 variant D225A expression This study pET22b-plu4D225N Vector for Plu4 variant D225N expression This study pET22b-plu4D25A Vector for Plu4 variant D25A expression This study pET22b-plu4D25N Vector for Plu4 variant D25N expression This study pET22b-plu4Y29H Vector for Plu4 variant Y29H expression This study pET22b-plu4Y29F Vector for Plu4 variant Y29F expression This study pET22b-plu4Y29R Vector for Plu4 variant Y29R expression This study 47 Strain/Vector Description Source pET22b-plu4C251A Vector for Plu4 variant C251A expression This study pET22b-plu4C251G Vector for Plu4 variant C251G expression This study pET22b-plu4C251N Vector for Plu4 variant C251N expression This study pET22b-plu4T189C Vector for Plu4 variant T189C expression This study pET22b-plu4T189S Vector for Plu4 variant T189S expression This study pET22b-plu4G26A Vector for Plu4 variant G26A expression This study pET22b-plu4A28T Vector for Plu4 variant A28T expression This study pET22b-plu4L117D Vector for Plu4 variant L117D expression This study pET22b-ind4G24A Vector for Ind4 variant G24A expression This study pET22b-ind4A26T Vector for Ind4 variant A26T expression This study 2.2.2 Cloning and mutagenesis The Plu4-containing plasmid (pET22b-plu4) was previously synthesized by Bio Basic Inc. and was sub-cloned into pET22b to contain a C-terminal histidine tag (unpublished). Plasmids from successful colonies were purified using the QIAprep Spin Miniprep Kit from Qiagen and transformed into chemically competent E. coli BL21 (DE3) by heat shocking for protein expression. pET22b-ind4 was made in a previous study.48 A description of the identification, cloning and characterization of other Ind4 homologs is described in Chapter 3. Plu4 and Ind4 residues targeted for mutagenesis were identified by analyzing crystal structures and sequence alignments made from known and predicted arginine hydroxylases and desaturases. Sequence alignments were made using Muscle in UGENE.175 The full sequence alignment was visualized with secondary structure information using ESPript 3.0.176 Plu4 and Ind4 variants were made using the primers listed in Table 2.2. Q5 polymerase (NEB) was used to amplify the template plasmid (pET22b-plu4 or pET22b-ind4) and introduce mutations by polymerase chain reaction (PCR). The PCR product was digested with DpnI to remove template DNA and transformed into E. coli DH5a. Colonies were miniprepped and sequenced using T7 primers (Table 2.2) to confirm that the gene had the intended mutation and was free of errors. Successfully mutated plasmids were transformed into chemically competent E. coli BL21 (DE3) by heat shocking for protein expression and purification. 48 Table 2.2: Oligonucleotide primers used in this study. Primer Sequence (5′ to 3′) Description T7 TAATACGACTCACTATAGGG T7 promoter for sequencing T7-term GCTAGTTATTGCTCAGCGG T7 terminator for sequencing Plu4-H27A-F2 CTTCGCAGACGGTGCGGCATACCATGACATCAACGAG H27A variant of Plu4 Plu4-H27A-R2 GATGTCATGGTATGCCGCACCGTCTGCGAAGTTGTACAGG H27A variant of Plu4 Plu4-D225A-F3 CTGGCCGACGCAAGCGCTGAAAATCTCCCTGATGGTG D225A variant of Plu4 Plu4-D225A-R3 GGAGATTTTCAGCGCTTGCGTCGGCCAGGTTTTACC D225A variant of Plu4 Plu4-D225N-F2 CTGGCCGACGCAAAACCTGAAAATCTCCCTGATGG D225N variant of Plu4 Plu4-D225N-R2 GGAGATTTTCAGGTTTTGCGTCGGCCAGG D225N variant of Plu4 Plu4-D25A-F CAACTTCGCAGCCGGTCATGCATACC D25A variant of Plu4 Plu4-D25A-R GGTATGCATGACCGGCTGCGAAGTTG D25A variant of Plu4 Plu4-D25N-F CAACTTCGCAAACGGTCATGCATACC D25N variant of Plu4 Plu4-D25N-R GGTATGCATGACCGTTTGCGAAGTTG D25N variant of Plu4 Plu4-Y29H-F CAGACGGTCATGCACATCATGACATCAACGAG Y29H variant of Plu4 Plu4-Y29H-R CTCGTTGATGTCATGATGTGCATGACCGTCTG Y29H variant of Plu4 Plu4-Y29F-F CAGACGGTCATGCATTCCATGACATCAAC Y29F variant of Plu4 Plu4-Y29F-R GTTGATGTCATGGAATGCATGACCGTCTG Y29F variant of Plu4 Plu4-Y29R-F CAGACGGTCATGCACGCCATGACATCAAC Y29R variant of Plu4 Plu4-Y29R-R GTTGATGTCATGGCGTGCATGACCGTCTG Y29R variant of Plu4 Plu4-C251A-F AAGAAATTTTCCTGGCGAGCAGCAACTTCGCC C251A variant of Plu4 Plu4-C251A-R GGCGAAGTTGCTGCTCGCCAGGAAAATTTCTT C251A variant of Plu4 Plu4-C251G-F AAGAAATTTTCCTGGGCAGCAGCAACTTC C251G variant of Plu4 Plu4-C251G-R GAAGTTGCTGCTGCCCAGGAAAATTTCTTC C251G variant of Plu4 Plu4-C251N-F AAGAAATTTTCCTGAACAGCAGCAACTTCGCC C251N variant of Plu4 Plu4-C251N-R GGCGAAGTTGCTGCTGTTCAGGAAAATTTCTT C251N variant of Plu4 Plu4-T189C-F CCTGCTGGACCGTTGCTTCCGTATCTACGG T189C variant of Plu4 Plu4-T189C-R CCGTAGATACGGAAGCAACGGTCCAGCAGG T189C variant of Plu4 Plu4-T189S-F CCTGCTGGACCGTAGCTTCCGTATCTACGG T189S variant of Plu4 Plu4-T189S-R CCGTAGATACGGAAGCTACGGTCCAGCAGG T189S variant of Plu4 Plu4-G26A-F CAACTTCGCAGACGCGCATGCATACCATGAC G26A variant of Plu4 Plu4-G26A-R GTCATGGTATGCATGCGCGTCTGCGAAGTTG G26A variant of Plu4 Ind4-G24A-F CTCGCCGATGCGCACGCCCATCAGGG G24A variant of Ind4 Ind4-G24A-R CCTGATGGGCGTGCGCATCGGCGAGATTG G24A variant of Ind4 Plu4-A28T-F CTTCGCAGACGGTCATACCTACCATGACATCAACG A28T variant of Plu4 Plu4-A28T-R CGTTGATGTCATGGTAGGTATGACCGTCTGCGAAG A28T variant of Plu4 Ind4-A26T-F GATGGCCACACCCATCAGGGCCAG A26T variant of Ind4 Ind4-A26T-R CTGATGGGTGTGGCCATCGGC A26T variant of Ind4 Plu4-L117D-F GATAACCTGTACGATCTGCTGAAACGTCGTGGTGTTG L117D variant of Plu4 Plu4-L117D-R GACGTTTCAGCAGATCGTACAGGTTATCAAAAGCCGGCTCG L117D variant of Plu4 49 2.2.3 Protein purification and Plu4/Ind4 variant characterization E. coli BL21 (DE3) cultures were inoculated from a 5 mL overnight culture into 1 L of Luria-Bertani (LB) media containing 100 µg/mL ampicillin at 37°C and 200 rpm to an OD600 of 0.4-0.6. The temperature and shaking were then lowered to 16°C and 150 rpm, respectively, for 30 min before addition of 0.5 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG). The cultures were incubated for 16 h at 16°C and 150 rpm before the cells were pelleted by centrifugation. Pellets were resuspended in a Buffer A (50 mM phosphate pH 8.0, 300 mM NaCl, 10 mM imidazole) and lysed by sonication. Cell lysates were clarified by centrifugation (40,500 x g for 45 min) to remove cell debris and diluted five-fold with Buffer A. The diluted supernatant was loaded onto Ni-charged Chelating Sepharose Fast-flow resin by gravity filtration. The resin was washed with five volumes of Buffer A, then with five volumes of Buffer B (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole). The protein was eluted from the resin with Buffer C (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM imidazole). Fractions containing the protein were yellow and their purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein-containing fractions were pooled and concentrated to ~6-8 mg/mL using an Amicon Ultra-15 Centrifugal Spin Filter (10 kDa molecular weight cut-off) from Millipore Sigma before being further purified on a HiLoad™ 16/600 Superdex™ 200 pg gel filtration column. The column was pre-equilibrated with Buffer D (20 mM HEPES pH 7.5, 50 mM NaCl) and was run at 1 mL/min to elute the protein. Fractions were pooled and all PLP and bound substrates were removed from Plu4 by dialyzing in Buffer D containing 10% glycerol and 50 mM of hydroxylamine for 2 h, then overnight into Buffer D containing 10% glycerol to remove hydroxylamine. Plu4 was reconstituted with PLP by dialysis overnight in Buffer D containing 10% glycerol PLP in five times molar excess of the protein concentration. The dialyzed samples were concentrated to ~40 mg/mL, as described above. The protein was aliquoted into 100 µL samples, frozen in liquid nitrogen and stored at −70°C. Protein concentrations were determined throughout purification by absorbance at 280 nm. Variants were purified on a smaller scale using the above method with small changes. Cells were lysed using BugBuster® 10X Protein Extraction Reagent (Novagen), diluted to working concentration in Buffer A. After centrifuging, the supernatant was diluted two-fold with Buffer A and purified using HisPur™ Ni-NTA Spin Column (Thermo Scientific), instead of the 50 Ni-charged Chelating Sepharose Fast-flow resin. The variants were not further purified by gel filtration and were dialyzed overnight in Buffer D containing 10% glycerol and PLP in five times molar excess of the protein concentration to reconstitute the PLP without first removing all bound PLP and substrates. Each Plu4 or Ind4 variant was assayed by electrospray ionization-mass spectrometry (ESI-MS). 100 µL reactions were set up overnight at 30°C with 1 mM L-arginine, 25 µg/mL catalase, 50 mM Tris-HCl (pH 7.5) and 5 μM of enzyme. Reactions were stopped with 100 µL methanol after 16 h and particulate matter was removed by centrifugation. A 20 µL aliquot of the supernatant was analyzed using a 6120 Quadrupole liquid chromatography-mass spectrometry (LC-MS) system (Agilent) operated in positive ion mode using a Poroshell 120 EC-C18, 2.7 µm, 4.6 x 50 mm column (Agilent). 2.2.4 Crystallization Initial crystals were obtained from 96-well screens, set up manually, using 5 and 15 mg/mL of protein. Diffraction-quality crystals were obtained from 2 µL of PLP-reconstituted Plu4 at 12-14 mg/mL mixed in equal amounts with the crystallization solution (0.1 M Bis-Tris:HCl, pH 6.5; 27% PEG 3350; 0.2 M ammonium acetate) in using hanging drop vapour diffusion methods. Spontaneous crystals were unable to grow at pH 6.5, so crystals were grown in the same condition at pH 6.0 and were used for seeding drops at pH 6.5. The seed stock was made by crushing a large, thin plate in a 7 µL drop of the mother liquor. Seeding was done by passing a seeding tool (Hampton Research) through the seed stock drop and transferring microseeds to a protein drop, which had been allowed to equilibrate over the well solution for 30 minutes. Crystals grew to diffraction quality after 3-7 days. Crystals were soaked by adding 1 µL of 10-15 mM of L-arginine and saturated PLP (~10 mM) in mother liquor or 1 µL of saturated PLP in mother liquor directly to the 4 µL crystallization drop, producing a final concentration of 2-3 mM L-arginine and/or ~2 mM PLP. Crystals were frozen when they were either yellow (2 h with PLP only) for the holo-enzyme structure, orange (8 minutes with PLP and L-arginine) for the intermediate-bound structure, or yellow (16 h with PLP and L-arginine) for the product-bound structure. Crystals were cryoprotected with 0.1 M Bis-Tris:HCl (pH 6.5), 28% PEG 3350, and 15% ethylene glycol prior to freezing in liquid nitrogen and collecting X-ray diffraction data. 51 2.2.5 Data collection, structure determination and computational methods Data were collected from the Stanford Synchrotron Radiation Lightsource (Menlo Park, California) at beamline 9-2 using a Dectris Pilatus 6M detector and a wavelength of 0.97946 Å. UV-Vis spectra were measured on each single crystal while mounted on the X-ray detector using a microspectrophotometer prior to collecting X-ray diffraction data sets. The microspectrophotometer uses UV solarization-resistant optical fibres, two reflective Newport Schwardchild objectives, Ocean Optics QE65000 Spectrum Analyzer and a Hamamatsu light source with deuterium and halogen lamps. The data set was scaled and integrated using autoxds. Plu4 crystallized as a homodimer in the space group P21, containing two or four monomers in the asymmetric unit. The data set was phased using Phaser-MR177 in the Phenix software package using Chain A of a RohP crystal structure (PDB: 6C3D)116 as a search model, which had 30% sequence identity to Plu4. The search model was generated using CHAINSAW178 in CCP4 to generate a poly-alanine model of the structure, without the N-terminus (residues 1-26 were removed). The initial model was built using Phenix Autobuild179. The structure was iteratively modified in COOT180 and refined with Phenix refine181 using translation-liberation-screw (TLS) refinement. Simulated annealing was also used during initial refinement iterations to remove model bias. Residues with side chains that did not fit the electron density were deleted. Missing residues and side chains were built in gradually according to the positive Fo-Fc density contoured at 3.0 σ. Active site molecules, including cofactor, substrate or other molecules from crystallization conditions, were also built in according to the positive Fo-Fc density contoured to 3.0 σ. Restraints for the non-standard molecules were generated in Phenix eLBOW.178 Ligands were built into the intermediate structure when refinement was nearly complete, including placement of water molecules. Although there is likely a mixture of intermediate ligands present in the active site of this structure, only Quinonoid I was built into the active site with full occupancy based on the dominant UV-Vis signal observed at 510 nm with the microspectrophotometer. Data collection information and refinement statistics are given in Table 2.3. Density functional theory (DFT) calculations were done to determine that the imine was the most stable tautomer form of the 4,5-dehydro-2-iminoarginine product independent of influence from the enzyme active site. All quantum mechanical calculations were performed with Gaussian 09.182 Geometry optimizations were calculated with the B3LYP183–186 density 52 functional with the IEFPCM model (diethyl ether),187 and the 6-31G(d) basis set for the imine and enamine system (Table 2.4). Free energy corrections were calculated using Truhlar’s quasiharmonic approximation.188,189 Single point energies were calculated using M06-2X190 with IEPPCM (diethyl ether),187 and the 6-311++G(d,p) basis set for the imine and enamine system (Table 2.5). Monte Carlo conformational searches were performed to identify lowest energy conformations with the OPLS3 force field191 in Maestro/Macromodel.192 Structure graphics were generated using CYLview.193 Docking of the more stable imine products were done through Autodock Vina194 to determine the likelihood of the observed product-binding mode in the product-bound structure. 53 Table 2.3: Data collection and refinement statistics. Holo Intermediate Product Data Collectiona Wavelength 0.9795 0.9795 0.9795 Space Group P21 P21 P21 Dimensions a 75.02 74.92 76.37 b 72.19 70.28 72.19 c 76.27 138.57 139.63 beta 112.09 98.11 98.44 Resolution (Å) 1.98 1.93 2.5 High Resolution Range (Å) 2.05 – 1.98 2.00 – 1.93 2.59 – 2.5 CC1/2b 0.997 (0.514) 0.996 (0.852) 0.995 (0.486) Completeness 98.6 (94.2) 98.8 (90.7) 99.1 (98.0) Unique Reflections 52155 (3510) 106646 (4801) 51795 (4444) Multiplicity 6.9 (6.6) 13.6 (12.3) 13.6 (13.1) I/σI 15.2 (1.4) 11.1 (1.9) 7.3 (1.5) Refinementa Rworkc 0.1663 (0.2753) 0.1512 (0.2334) 0.2339 (0.3804) Rfreed 0.2159 (0.3241) 0.1944 (0.2900) 0.2711 (0.4526) No. non-hydrogen atoms 6411 13549 11779 Protein 6029 12130 11503 Solvent 330 1130 180 Ligands 52 289 96 RMSD bonds (Å) 0.010 0.012 0.007 RMSD angles (deg) 1.31 1.51 0.83 Ramachadran favoured (%) 97.45 97.94 96.84 Ramchadran allowed (%) 2.55 2.06 3.16 Ramachadran outliers (%) 0 0 0 Average B-factor (Å2) 52.1 30.6 67.3 Protein 51.8 29.6 67.4 Solvent 52.1 39.3 54.7 Ligands 80.1 41 82.4 No. TLS groups 23 38 27 aValues in outermost resolution shell are given in parentheses bCC1/2= ∑ (𝑥! − ?̅?)(𝑦! − 𝑦()! /*∑ (𝑥! − ?̅?)"! ∑ (𝑦! − 𝑦()"! for a random half of unique reflection measurements cRwork= ∑ +|𝐹#(ℎ𝑘𝑙)| − |𝐹$(ℎ𝑘𝑙)|+%&' /∑ |𝐹#(ℎ𝑘𝑙)|%&' for the 95% of reflection data used in refinement dRfree is calculated identically to Rwork, but only for the 5% of reflection data not used in refinement 54 Table 2.4: Cartesian coordinates of optimized structures for DFT calculations of imine and enamine tautomer stabilities. N -0.843153 -2.576308 -0.254737 C -0.538314 -1.511212 0.397897 C -1.041715 -1.226064 1.776727 C 0.440129 -0.545243 -0.378946 C -1.768424 0.098559 1.812864 O 0.710692 -0.966771 -1.522319 C -3.046291 0.21358 2.178174 N -3.683775 1.469455 2.291029 H -3.089864 2.291223 2.291936 C -5.010863 1.675031 2.359907 N -5.864144 0.691593 2.053299 N -5.46707 2.864678 2.760479 H -5.587259 -0.060723 1.437834 H -6.854172 0.796412 2.228491 H -4.860876 3.535247 3.212811 H -6.434988 3.125991 2.632134 H -1.44156 -3.324479 0.087095 O 0.783508 0.459293 0.267258 H -1.668014 -2.042464 2.150647 H -1.173268 0.960867 1.526783 H -3.66011 -0.633075 2.467842 H -0.150708 -1.157087 2.417263 H -0.408746 -2.620687 -1.188919 N -0.000203 -2.599957 0.314223 H 0.394569 -2.250783 -0.657646 C -0.872337 -1.430574 0.545575 C -1.649166 -1.328627 1.637326 C -0.686144 -0.485783 -0.654518 C -2.509257 -0.192796 1.892036 O 0.141375 -0.970294 -1.497975 C -3.298118 -0.120622 2.978247 N -4.168412 0.962533 3.189361 H -4.280566 1.616697 2.421712 C -4.848678 1.231394 4.32028 N -4.520724 0.629356 5.468384 N -5.867909 2.093464 4.275392 H -3.586774 0.273491 5.619096 H -5.117055 0.709272 6.281154 H -6.295045 2.360646 3.398721 H -6.257455 2.488264 5.12047 H 0.748815 -2.712195 1.003066 O -1.313579 0.582243 -0.690791 H -1.641298 -2.13068 2.375381 H -2.48591 0.595824 1.144365 H -3.349444 -0.905335 3.725405 H -0.49822 -3.489732 0.221317 Table 2.5: Energies of imine and enamine tautomers. ZPVE = zero-point vibrational energy; TCE = thermal correction to energy; TCH = thermal correction to enthalpy; TCG = thermal correction to Gibbs free energy. Energies are given in kcal/mol. ZPVE TCE TCH ECG E H (E+TCH) G (E+TCG) imine 0.188227 0.201426 0.202370 0.147147 -604.539284 -604.336914 -604.392137 enamine 0.187753 0.200762 0.201706 0.148001 -604.334089 -604.333145 -604.386850 OONHH2NNH2NH2OONHH2NNH2NH355 2.3 Results 2.3.1 Plu4 crystal structures To elucidate the determinants of catalysis in arginine desaturases, attempts were made to solve the X-ray crystal structure of Ind4. However, crystals could not be obtained for Ind4, so Ind4 homologs were cloned and purified to obtain crystal structures of various points in the enzyme’s catalytic cycle. Plu4, an Ind4 homolog (32% sequence identity) from a convergent indolmycin gene cluster in P. luteoviolacea,68 was the only homolog that produced crystal structures with trapped intermediates and products. By freezing the crystals soaked under aerobic conditions for different periods of time coinciding with colour changes in the crystal (Figure 2.2), three crystal structures were obtained: holo-enzyme (yellow), intermediate-bound enzyme (orange) and product-bound enzyme (yellow after previously being orange) (Figure 2.3). Each crystal structure represents a different stage of catalysis and can be compared to the hydroxylase crystal structures of RohP and SwMppP. Holo2 mM PLP2 hoursIntermediate2 mM PLP and 3 mM Arg8 minutesProduct2 mM PLP and 2 mM Arg16 hoursFigure 2.2: Plu4 crystals after soaking PLP or PLP and arginine at different time points. 56 2.3.1.1 Holo-enzyme structure The holo-enzyme structure was obtained by soaking with 2 mM PLP for 2 h and the structure was solved to 2.0 Å resolution (Figure 2.3a). This structure was a homo-dimer with two molecules in the asymmetric unit. The overall fold of this structure matches the hydroxylases and other fold-type I PLP-enzymes195,196 with root-mean-square deviations (RMSDs) of 1.184 Å (523 atoms) when compared to MppP-5DJ1114 and 1.474 Å (622 atoms) 0.70.80.911.11.21.3350 450 550 650Absorbance (A.U.)Wavelength (nm)0.40.50.60.70.80.911.11.2350 450 550 650Absorbance (A.U.)Wavelength (nm)0.50.550.60.650.70.750.80.85350 450 550 650Absorbance (A.U.)Wavelength (nm)abcdefNHOH2-O3PONLys219NHOH2-O3PONOONHH2NNH2NHOONHH2NNH2NHOH2-O3PONLys219Figure 2.3: X-ray crystal structures of Plu4. (a) Holo-enzyme. (b) Quinonoid I structure. (c) Product 2-bound structure. The left image shows the overall fold, the middle panel shows the active-site view, and the right panel shows microspectrophotometer UV-Vis spectra obtained from the crystal used for X-ray crystallographic data collection and the corresponding molecules built into the active site. The PLP cofactor is shown with magenta carbons. 57 when compared to RohP-6C3B116 using Ca alignments (Table 2.6). Each monomer in the dimer contained three domains: a large domain (residues Asp31-Phe270), a small domain (residues Gly271-Lys381) and the N-terminal domain (residues Met1-His30). The large domain consists of 6 parallel b-strands and 1 antiparallel b-strand and 10 a-helixes arranged into an a-b-a sandwich. The small domain contains two antiparallel b-strands and three a-helices. The N-terminal domain consists of one a-helix and is located at the dimer interface, helping to enclose the active site. Unlike the RohP and MppP holo-enzyme structures, where the N-terminal regions are disordered or pointed completely away from the active site, the N-terminal region of the Plu4 holo-enzyme structure is fully ordered and covers the active site (Figure 2.4). The active sites are found between the large and small domains within the same monomer with additional contributions from some residues of the large domain of the other protomer in the dimer. Table 2.6: RMSD values for Plu4 holo-enzyme, intermediate-bound, and product-bound structures compared with RohP and SwMppP structures. The RohP Quinonoid I value is represented as QI, while Quinonoid II is represented as QII. The SwMppP intermediate was an external aldimine. RMSDs are from Ca alignments and are given in Å with the number of atoms included in each alignment in brackets. RohP SwMppP Holo 1.474 (622 atoms) 1.184 (523 atoms) Intermediate QI: 1.456 (621 atoms); QII: 1.428 (657 atoms) 1.12 (529 atoms) Product 1.476 (667 atoms) 1.153 (571 atoms) The UV-Vis data collected from the holo-enzyme crystal showed a characteristic 415 nm peak for the Schiff base (Figure 2.3a).197 Therefore, an internal aldimine was built into the holo-enzyme active site attached to Lys219 via a Schiff base. The PLP cofactor is also held in place by other residues characteristic of the fold-type I PLP-enzymes. Asp187 interacts with the nitrogen group of the PLP-pyridine ring, modulating the electrophilic properties of the PLP cofactor (Figure 2.5b).196 Asn159, found at the C-terminal end of the 4th b-strand, interacts with the phenolic oxygen and is common among PLP-dependent aminotransferases, but is not invariant.198 Additionally, the phosphate group of PLP interacts with the Ala87 backbone nitrogen and the side chains of Ser88, Lys227, and His27 (Figure 2.5b). While the identity and conformation of most of the active site residues are identical between the RohP, SwMppP, and Plu4 holo-enzyme structures, some residues are in slightly different conformations. His27 in 58 Plu4 matches the conformation of His34 in RohP, but His29 in SwMppP has two observed conformations (Figure 2.5c). Phe112 in Plu4 appears in a similar conformation to Phe115 in MppP, but the corresponding residue (Phe119) has rotated ~120° in RohP (Figure 2.5d). As a result of this rotation, the conformations of Asn121 and Leu122 in RohP have also shifted in comparison to the corresponding Asn114 and Leu115 in Plu4. Finally, Phe190 in Plu4 is rotated ~90° compared to Phe191 and Phe201 in MppP and RohP, respectively (Figure 2.5e). abcFigure 2.4: Comparison of holo-enzyme overall structures for (a) Plu4, (b) RohP (PDB: 6C3B), and (c) SwMppP (PDB: 5DJ1). The N-terminal regions, if present, are shown in yellow. 59 bF112F119F115N114N117N121L115I118L122daF190F201F191eH34H27H29D227D225D32D25D27D241cT189C200S190D188D187D198fFigure 2.5: Plu4 Holo-enzyme active site analysis. (a) Fo-Fc omit map is shown for the PLP cofactor contoured to 3σ. The PLP internal aldimine cofactor is shown in magenta. (b) Polar interactions between the enzyme and the cofactor are indicated with dashed lines and the corresponding distance in Å. The cofactor is shown in magenta. (c), (d), (e) and (f) shows a comparison of Plu4, RohP (PDB: 6C3B; blue) and SwMppP (PDB: 5DJ1; plum) active site residues, with cofactors shown in light green for Plu4, light blue for RohP and pink for SwMppP. Residue identities and numbers are given in the colour of the corresponding structure. Structures were aligned using the superpose function in PyMOL using Ca pairs. 60 2.3.1.2 Intermediate-bound structure An intermediate-bound structure of Plu4 was obtained to 1.9 Å resolution by soaking a crystal with 3 mM of 1 and 2 mM of PLP for 8 minutes, during which the crystal changed from yellow to orange. This structure, similar to the holo-enzyme structure, was a homodimer but it consisted of four molecules in the asymmetric unit, rather than two. The UV-Vis data obtained from the microspectrophotometer measurement of the orange crystal showed a major peak absorbance at λmax 510 nm and a minor peak at λmax 578 nm (Figure 2.3b). These peaks were similar to UV-Vis data obtained for Ind4, where a peak at λmax 511 nm was assigned as Quinonoid I and a peak at λmax 567 nm was assigned as Quinonoid II during stopped-flow experiments.113 When refinement was nearly complete, including placement of water molecules, five possible intermediates were built into the active site density (Figure 2.6). Although the presence of major and minor peaks in the UV-Vis microspectrophotometer data indicates that there is likely a mixture of intermediates present in the intermediate-bound structure, Quinonoid I was chosen as the ligand for the final structure based on the dominant UV-Vis signal observed at 510 nm. While fold-type I PLP-dependent enzymes can undergo large structural changes upon ligand binding,199,200 no major changes to the domains were observed between the holo-enzyme structure and the intermediate-bound structure. Unlike RohP and SwMppP, whose N-terminal helices become ordered upon intermediate formation, the N-terminus of Plu4 did not change conformation. Despite the N-termini appearing in similar, more ordered states, for the RohP, SwMppP and Plu4 intermediate-bound structures, Plu4 remains distinguished from the hydroxylases by having lower B-factor values assigned to the atoms in its N-terminus (Figure 2.7). The lower B-factor values indicate that there could be less N-terminal movement in Plu4 than in RohP or SwMppP. However, differences in the N-terminal helix could potentially be the result of crystallographic contacts between asymmetric units because the area surrounding the RohP and SwMppP N-terminal helices is more open than for Plu4, likely contributing to a more ordered N-terminal helix in Plu4 (Figure 2.7). 61 Many of the contacts between the internal aldimine of the holo-enzyme structure and the Quinonoid I ligand of the intermediate-bound structure remain similar; however, the PLP cofactor rotates away from Lys219 by 23.8° (Figure 2.8a and b), anchored by the heterocyclic nitrogen and phosphate group. Additionally, the bond between carbon and oxygen from the phosphate group rotates to accommodate this movement (Figure 2.8a). As the Quinonoid I intermediate is formed, Lys219 is released from the imine bond and forms a polar interaction with His27, Asp216 and the PLP phosphate group, facilitated by a water molecule (Figure 2.8c). With the addition of substrate, several new polar contacts are formed between the Quinonoid I intermediate and the enzyme. The anionic carboxylate group from Quinonoid I forms an ionic interaction with the positively charged Arg354 guanidinium group, which is a conserved residue in the PLP aminotransferases198. The carboxylate group from Quinonoid I also forms a complex hydrogen bonding network between Asn159 and Glu13 (Figure 2.8d). The guanidinium portion NHH2NNH2NOONHOH2-O3PONHH2NNH2NOONHOH2-O3PONHH2NNH2NOONHOH2-O3PONHH2NNH2NOONHOH2-O3PONHH2NNH2NOONHOH2-O3POQuinonoid IEnamine I Quinonoid IIEnamine IIExternal aldimineFigure 2.6: Comparison of possible intermediates modeled into the active site density for the intermediate-bound structure. Fo-Fc electron density maps are shown in green and contoured to 3σ. 62 of the Quinonoid I ligand interacts with a phosphate oxygen atom from PLP and the Ser88 side chain as well as with Thr10, found in the N-terminal helix and backbone oxygens from Glu246 and Glu247, found in the other protomer of the dimer (Figure 2.8e). Additionally, both His27 and Glu13 are both within 4 Å of C4 and C5 of the Quinonoid I intermediate and could serve as the catalytic residues responsible for deprotonating these carbons during catalysis (Figure 2.8f). His27 also has several nearby residues, including Asp225, Tyr29, and Cys251 that could be aiding His27 in any potential catalytic role (Figure 2.8f and Figure 2.9b). All other residues surrounding the active site appear to be unchanged in response to formation of Quinonoid I (Figure 2.8a). 63 abcFigure 2.7: Comparison of N-termini in intermediate-bound structures, (a) Plu4, (b) RohP (PDB: 6C3D), and (c) SwMppP (PDB: 6C8T). The left panel shows B-factors for the respective structures. B-factors are indicated by a spectrum of blue, white and red with blue having the lowest B-factors (below 20 Å2) and red having the highest B-factors (above 50 Å2). The N-terminal helices of both monomers are indicated with a black circle. The right panel shows N-termini (blue) for the respective structure (green) with surrounding asymmetric units (cyan/magenta/yellow/lightpink). Differences to the B-factors of the N-terminal helices could be due to crystal contacts (shown in the right panel) and would require a further comparison of the molecular dynamics of each protein to elucidate the role of the N-termini. 64 ac de fC4C5bFigure 2.8: Plu4 intermediate ligand interactions with protein in the intermediate-bound structure (a) Comparison of holo (green), intermediate (teal) and product (purple) structures for Plu4 with their respective ligands/cofactors shown in light green, light teal, and deep purple. (b) Comparison of holo internal aldimine (light green) and intermediate quinonoid I (light teal) structures with angle of movement given in degrees. (c) Interactions between Lys219 and surrounding His27, water, Asp216, Lys227 and the PLP phosphate group. (d) and (e) show polar contacts between the intermediate and the protein in Å. (f) Distances between carbons 4 and 5 and nearby His27 and Glu13 residues. Polar distances between His27 and Asp225 in the catalytic dyad are also shown. 65 2.3.1.3 Product-bound structure3 The product-bound structure of Plu4 was determined to 2.5 Å resolution by soaking a Plu4 crystal with 2 mM PLP and 2 mM 1 for 16 h, during which the crystal changed from yellow to orange and back to yellow. There were four molecules in the asymmetric unit, each forming half of a homodimer. The UV-Vis spectra obtained from this crystal had a peak at 413 nm, indicating the presence of an internal aldimine, which was subsequently built into the structure (Figure 2.3c). There were no large structural changes observed in this structure compared to the holo-structure or the intermediate structure. However, one or two segments of an external loop (Asp302-Glu303 in Chain A and Gly293-Thr294 and Asp302-Glu303 in Chain B) were 3 DFT calculations in this section were completed by M.G.-B. H27H34H29C251N267G253D225D241D227Y29H36R31D25D32D27bF119F112F115I118L122L115N114N121N117cF190F191F201K235K219K221dE13 E20E15T10T12T17aFigure 2.9: Comparison of Plu4, RohP (blue; PDB: 6C3D) and SwMppP (plum; 6C8T) intermediate-bound active site structures. The intermediate ligands are show in cyan, light blue and pink, respectively for Plu4, RohP and SwMppP. Residue identities and numbers are given in the colour of the corresponding structure. Structures were aligned using the superpose function in PyMOL using Ca pairs from Chain A. 66 disordered in the product-bound structure but were ordered in the holo-enzyme and intermediate-bound structures. There were two active sites that displayed electron density corresponding to a product, one in each dimer in the asymmetric unit. However, before modelling any products in this density, DFT model calculations were performed to determine which tautomeric form of the four-electron oxidized product was the most stable: the enamine or the imine structure. Surprisingly, the imine form was calculated to be the most stable, despite the enamine form being more conjugated (Figure 2.10). The imine form is likely to be more stable than the enamine because the distal ends of the conjugated system have highly electrophilic functional groups (guanidine and carboxylate), which may pull electron density away from the middle of the molecule, allowing the conjugation to break and the imine to form. Additionally, when bound to the enzyme, residues surrounding the guanidine and carboxylate may be acting to increase the electrophilicity of these electrophilic functional groups. Therefore, the imine form of 2 (Figure 2.1) was modelled into Chain A, where it fit well into the density, but 4 (Figure 2.1) was modelled into the active site of Chain C because it fit the electron density better (Figure 2.11). Although 2-iminoarginine is expected to be a product of Plu4 (Figure 2.1), its production has not been confirmed, so 4 was modelled instead. kcal/mol ∆E ∆Gimine 0.0 0.0enamine 3.0 3.6OONH2NHH2NNH2OONH3NHH2NNH2Figure 2.10: DFT calculations for four electron oxidized product in imine and enamine forms. 67 Each product was modelled into the active site with the guanidine group adjacent to the phosphate group of PLP, while the carboxylate group formed a salt bridge with Arg354 (Figure 2.11 and Figure 2.12), similar to what was observed in the RohP and SwMppP product-bound structures. However, the imine of 5 (Figure 2.1) and the ketone of 4 (Figure 2.1) were pointed toward the carboxylate group of Glu13 rather than towards the 4′-carbon of PLP. The latter was the expected conformation of the products considering that the intermediate ligand requires that the imine nitrogen be bonded to the 4′-carbon of PLP prior to reformation of the internal aldimine. The RohP and SwMppP product-bound structures both showed the ketone group of 3 pointed towards the 4′-carbon of PLP in the expected conformation (Figure 2.12c and Figure 2.13a). To determine if this unexpected binding conformation observed in the Plu4 product-a bc dFigure 2.11: Electron density for products 2 and 4 in the product-bound structure. (a) Fo-Fc omit map density for 2 (salmon) in Chain A of the product bound structure. (b) Fo-Fc omit map density for 4 (salmon) in Chain C of the product bound structure. (c) 2Fo-Fc and Fo-Fc of 2 when fit into the Chain C active site. (d) 2Fo-Fc and Fo-Fc of 4 when fit into Chain C active site. Fo-Fc maps are contoured to 3s and 2Fo-Fc maps are contoured to 1s. 68 bound structure is reasonable, docking experiments were done to determine energetic feasibility of the observed binding conformations for both modelled products, 4 and 5. The docking studies determined that both binding conformations, with the imine/ketone pointing either toward Glu13 or the 4′-carbon of PLP are energetically feasible (Figure 2.12). a bc-6.4 kcal/mol-5.1 kcal/moldFigure 2.12: Analysis of product binding conformations. (a) Polar interactions of the carboxylate and imino groups of 2 (salmon) in Chain A. (b) Polar interactions of the carboxylate and ketone groups of 4 (salmon) in Chain C. (c) Polar interactions between the carboxylate group of 3 (salmon) and active site residues in RohP (PDB: 6C3A). The distance between the ketone of 3 and 4′-carbon of the PLP cofactor is also shown. (d) Docking conformations (green) closely matching observed conformations in the Plu4 product-bound crystal structure (cyan) for 2 (top) and 4 (bottom) with binding energy indicated below. 69 2.3.2 Site-directed mutagenesis Guided by crystallographic studies of Plu4 and previous mutagenesis work on the PLP-dependent arginine oxidases, it was hypothesized that His27 and Asp225 in the active site could play a role in the second oxidative step. Both the His27 and Asp225 residues are highly conserved in the PLP-dependent arginine oxidases (Figure 2.14) and the His residue has been implicated previously, through mutagenesis, as a catalytic residue for installing the 4-hydroxyl group of 3 (Figure 2.1) in RohP.116 To test the effect that conserved His and Asp residues may have on catalysis in Plu4, His27 and nearby conserved residues Asp225 and Asp25 were mutated. In the H27A, D225A, D225N, and D25A variants, the first oxidation product 4 (Figure 2.1) is still made, but the second oxidation product 5 is not (Table 2.7). D25N had the same E13E20E15T10T12T17aH27H34H29C251N267G253D225D241D227 Y29H36R31D25D32D27bF190F191F201dF119F112F115I118L122L115N114 N121N117cFigure 2.13: Comparison of Plu4, RohP (blue; PDB: 6C3A) and SwMppP (plum; 6C9B) product-bound active site structures. The product ligands and PLP are shown in deep purple, light blue and pink, respectively for Plu4, RohP and SwMppP. Residue identities and numbers are given in the colour of the corresponding structure. Structures were aligned using the superpose function in PyMOL using Ca pairs. 70 product profile as the wild-type enzyme (Table 2.7), suggesting that Asp25 does not have an essential role in catalysis. Table 2.7: Product profiles of enzyme variants based on LC-MS data. Variant Enzyme Products Made WT Plu4 4 and 5 H27A Plu4 4 D225A Plu4 4 D225N Plu4 4 D25A Plu4 4 D25N Plu4 4 and 5 Y29H Plu4 4 and 5 Y29F Plu4 none Plu4S-31Psy4Ind4Bfl4Pti4RohPSeuPscSwMppPSMOE7SlyF A D G H A Y HL A D G H A Y QL A D G H A Y HL A D G H A H QL A D G H A H QV A D G H A H QL A D A H T H QL A D A H T H QL A D A H T H QI A D G H A R QI A D G H A R QI A D G H A R Q232318212124183634252525303025282831254341323232I E D T G K T W P T Q D L K IF E D T G K I F P T Q D M K AF E D T G K T W P T Q D L K AV E D T G K T W P T Q D L K CV E D T G K T W P T Q D L K CV E D T G K T W P T Q D L K CI E D T G K T W P V Q D A K CI E D T G K T W P V Q D A K CM E D T G K T W P L Q D T K CI E D T G K L W P T L D L K AI E D T G K L W P T L D L K VI E D T G K L W P T L D L K V214212212212212215218236235216216216228226226226226229232250249230230230....................................Figure 2.14: Alignment of sequences from characterized SSN groups (I-III). Group Ia is shown in blue, Group Ib is shown in yellow, Group II is shown in green, and Group III is shown in purple. Known catalytic residues are indicated with a filled black arrow, while Asp225, which participates in a catalytic dyad, is shown with a hollow black arrow. Sequences shown are abbreviated as follows: Plu4 represents WP_039608190.1 from Pseudoalteromonas luteoviolacea, S-31 represents WP_030739174.1 from Streptomyces sp. NRRL S-31, Psy4 represents SDZ12441.1 from Pseudomonas syringae strain BS2900, Ind4 represents AJT38685.1 from Streptomyces griseus subsp. griseus, Bfl4 represents WP_061786414.1 from Bacillus flexus, Pti4 represents WP_143006797.1 from Paenibacillus tianmuensis, RohP represents WP_041824998.1 from Streptomyces cattleya, Seu represents WP_102919045.1 from Streptomyces eurocidicus, Psc represents WP_046237671.1 from Pseudomonas syringae pv. Coryli, SwMppP represents the sequence from PDB ID: 5DJ3_A from Streptomyces wadayamensis, SMOE7 represents ARH92424.1 from Streptomyces sp. MOE7, and Sly represents AOP50599.1 from Streptomyces lydicus. 71 Variant Enzyme Products Made Y29R Plu4 4 and 5 C251A Plu4 4 and 5 C251G Plu4 none C251N Plu4 4 and 5 T189C Plu4 4 and 5 T189S Plu4 4 and 5 T186C Ind4 none G26A Plu4 none G24A Ind4 none A28T Plu4 none A26T Ind4 none L117D Plu4 none Next, residues within 5 Å of the active site in Plu4 that differ between the hydroxylases and the desaturases were targeted to try to find the cause of the different reactivities (Figure 2.15). These residues include Thr189, Gly26, and Ala28, each of which has a different identity to Plu4 in both RohP and SwMppP. The corresponding mutations were also made in Ind4 to match the RohP identity. Substitution of G26A and A28T in Plu4 showed complete loss of both products, but T189C and T189S in Plu4 gave no change in the product profile (Table 2.7). However, all mutations at these positions in Ind4 resulted in loss of both products (Table 2.7). Two non-conserved residues, Tyr29 and Cys251, near Asp225 were also targeted to see if they changed the product profile, but the resulting variants either abolished production of both 4 and 5 or did not affection production (Table 2.7). These residues seem likely involved in maintaining structure around the catalytic residues, rather than determining hydroxylase and desaturase reactivity. Leu117, which is in a hydrophobic tunnel leading to the active site in the Plu4 structure, was also targeted. This tunnel is far less hydrophobic in the RohP structure, seemingly as a result of an aspartate residue at the position of Leu117. However, no products were observed from this variant (Table 2.7). 72 Plu4 1 10 20 30 40 50 60 70 Plu4 T E N AD H I L SL L G A N I W E F F LI S.............MNTDRYD EV VEG SYLY F YHDI EHYVD NN QRY QEGKDHSIPDM KA KNQ AE DS-31 T E N AD H I L TL T L G A Q T Q V G AW E E Y Y LI S.............MGDQYVK DY QIGFL EA Y GL AG EV DR N RKS AESIPES RR IDR AE GPsy4 T E N AD H I L TL L G A M IW D F F LI S..................MN DV WNAFALPA YHDMHVAFME KN ER SEA KITPPRMALD KNS SR KInd4 T E N AD H I L NL I Q L G AHQ N Q V P IF E E F FY LA Q...............MERFN RY HVG N AI GQ ST QA AD N EAA NALQATT RE QRA T GBfl4 T E N AD H I L NL L Q L G AHQ S Q I IF E E F FF LA Q...............MTKFN RY SVG N DF GQ ET KK SE SN YNS LTKQKAL EE QNE K EPti4 T E N AD H I L NL I Q V G AHQ S Q I P LF E E F FY LA Q............MKSLMRMP RY SIG N LF PQ DA RQ GR D DQA TARQAEL DE RRL T GRohP T E N AD H I L L L L HQ S Q V P LW E E F FF L Q..................MLD QH IQA TMKY A T RQ AS QS SR Q YEA EGLQATY KR TEA Q HRSeu T E N AD H I L L L T L HQ S Q V P LW E E F FF L QMTPHGTTAGPGTAQGSGLLPD QH IQA R RF A T RQ AA RG ER E YEA NGLQATY QR TEA R HRPsc T E N AD H I L I L T L HQ S Q V P LW E F FF Q..MNSLYAGIDTLSAVPVLDD QH ISA K RF A T DQ ET RM AS E YRAQNQTQYQS QA IQA TFHGSMOE7 T E N AD H I L NL L S I G ARQ T Q V P LF E E FF AL Q...........MGESEGDTG QL FLA N EF AL AG SK DD L AEG KRPVEEL QEAHAA T GSly T E N AD H I L NL L S I G ARQ T Q V P LF E E FF AL Q...........MGESEGDTG QL FLA N EF AL AG SK DD L AEG KRPVEEL QEAHAA T GSwMppP T E N AD H I L NL L S I G ARQ S Q V P LW E E YF LL Q...........MTTQPQLKE QW YLA N EL AL PG QK NE V AES QRPVQQI SEAHQA T G SwMppP . Plu4 80 90 100 110 120 130 140 150 Plu4 S L L P FDN L E TL TA S DIV W I L L K V I L V DAS HSTNHFS.VCP N I AA HKENKRTA E A YL RRG D SAFD LALKN...EHQ AQI S..SGDIS-31 S L L P FDN L E L L TA S DLV Y V L I R A PV L L DVAG RAAPGH .ILP N I AA TLTGKR L TR T AL RRG DPS S REISD...GES ARR P....GTPsy4 S L L P FDN L E SL SA S DLI F V L L V V V EAP MPHTHYK.ICP N I AA HGKNIRTV E T AL QRRG T DS S DNLRRHLVDGNFDSIPA..IKTAInd4 S L L P FDN L E SA M YSA LS DLV F V L L I R V V PL L DAH VDHPRT .LC T AT ASRNLT G Q C AT RRQ K V G EQFR....PES DRTFA..ELNTBfl4 S L L P FDN L E SA M YSA LS DLI Y V L L I K V L PV I L DAF TNHPRT .LC T AT QKNKLS S Q C AT RRD H V A DRFT....PSN LHT D..NMTTPti4 S L L P FDN L E TA L YSA LS DLI Y L L L V L PL M DAP AGLTRT .FC T AT ASRGLTTA H C AT QRRN A T H EELR....LDR EETFA..GLAARohP S L L P FDN L E TA M Y A IS VA F V I L V V L PI I L DAP LVKNKT .LS A TM GM KKERLA T E C YD ANMD P Y D SVFYD...VDR YPE ER.RVRTSeu S L L P FDN L E TA L Y A IS VA Y V I L V R V L PI L L DAP LAKNKT .LS A TM GM KKQRMD T E C YD NMD P Y D AVLHD...PDS YER RR.QVRTPsc S L L P FDN L E A I Y A I I Y V I L L K V M L V L DAHH LKRHDE YLV A AMH TAT RKKNMS G E C HD HIE P SA D SLFTS...STE YAK QKAAIGLSMOE7 S L L P FDN L E S V YS V EIL V V I L R L L PL L LH YPSAPGR LSC S AM SRS AETIDT A H T AD GNG K V A DPLH....GDD DAS ...LASVGCSly S L L P FDN L E S V YS V EIL V V I L R L L PL L LH YPSAPGR LSC S AM SRS AETIDT A H T AD GNG K V A DPLH....GDD DAS ...LASVGCSwMppP S L L P FDN L E V YS VS EIL V V I L R L L PV L LHGYPAEPGR LSC S M ARS SASVDR A H T AD GNG D V E DALH....GAD SAE ...LSSVGC TT .... ... SwMppP TTT TT Plu4 160 170 180 190 200 210 220 Plu4 F PNNPTG C D F D Y L EDTGK P D K L L M LV ILL T R Y F I SGI YVVI W TQ L VN LE ...TESEFVY EQ KAHNIT R I GKTN..... D Q EQ D T IS-31 F PNNPTG C D F D Y L EDTGK P D K L L L LV IVI S R F W V GA YIV F TQ M VN TN ...SEEVFTA RG VEHAVT A L HPQT..... A R QDL S F I APsy4 F PNNPTG C D F D Y L EDTGK P D K V I L LV LLVV S R F Y V SGV FIA W TQ L VS YT ...SLQEFKT AY KRTEK N F NRQG..... D Q LE N F T AInd4 F PNNPTG C D F D Y L EDTGK P D K V L L VI ILIV T R F W SGI YVVV W TQ L TL FH ...DQEQFRR DL AEHQK C F APTPF.... Q AQ EE S T CBfl4 F PNNPTG C D F D Y L EDTGK P D K L L L VI LLII T R Y W A SGV Y MV W TQ L TL FM ...SQDEVKT KH KENDK C F HQDSF.... Q E ED S F T CPti4 F PNNPTG C D F D Y L EDTGK P D K V V L LV LLIV T R F W L S M YLMV W TQ L TL FE ...SREEWER AM GQYGK C F SRKPL.... Q E EK N S T CRohP F PNNPTG C D F D Y L EDTGK P D K L L L VV LLLI F F L SGV YLAI W Q A VD FS LRHGRKGFEE RF KDHDK FC AS TLFEPELAR M E EN R T V CSeu F PNNPTG C D F D Y L EDTGK P D K L L L VV LLLI F F L SGV YLAI W Q A VD FS LKHGRKGFEE RF KDHDK FC AA TLFDPEVAR V E EN R T V CPsc F PNNPTG C D F D Y L EDTGK P D K I L L VA LLIL F I G YLVM W Q VD SS FAEGPDTFVE RY RDYNK FC AS .FKAAGKHRV A A DDI TQ T L T CSMOE7 F PNNPTG C D F D Y L EDTGK P D K V L V LA ILAL S R F Y V SGV WVVI W T L TT RV ...SKERLTR EQ AAAGV T G DTRAH.... H E EA R L L VSly F PNNPTG C D F D Y L EDTGK P D K V L V LA ILAL S R F Y V SGV WVVI W T L TT RV ...SKERLTR EQ AAAGV T G DTRAH.... H E QA R L L VSwMppP F PNNPTG C D F D Y L EDTGK P D K V V L LA VLAL S R F Y V G WVVI W T L TT RV ...AEERLRR EQ AEHGT T G DAAAH.... H A QEA CR L L A TTT TT SwMppP Plu4 230 240 250 260 270 280 290 300 310 Plu4 S L S L LMV E I M L EI FA LL F GV I V I L G L L L WS Y A SST RL YE F CS N A KQ VAVTAKF DAT KNE RRRSET NDA A .TG KVFDNDEKCQ P C IS-31 S L S L LL L I NEI V F VL F E GL V V R LR L V ISV WS SC AEHRAP RE Y Y C K T R GD FE ARVT HRS HSL ARR TR QA GE.DL EREARS..SR E LPsy4 S L S L LII D V L NEI V FV L GL L V NR LR L G L LSV WS Y T KVFFES Y Y C N H FSHLFDATSAV KSV WDV DS AL ET Q .SV RISETSKYSL E LInd4 S L S L ILA L V L NDIL V PFI LL Y D GL I V NR LR L G L ISV WS V TD HES LE H N Q TA LR TERN QQT WSV RA IS KA E .TV VPAHPE..ST E VBfl4 S L S L ILA E L I L NDIL V PFI LL Y D GI L I NR LR I ISV WS A D YEG LE H N N IE IK AQKV SSS WKV EE TY NV KD.SIFVPHSDD..TM E LPti4 S L S L ILA E L V L NDIL V PFI LL Y D GL I I NR LR I L ISV WS M D ADD LE H N Q IE LK TARH QSV WET DR KT EA RS.SG VLNNPG..ST E LRohP S L S L LI D I V L T VL V PFV ML Y D L V L NR AR L G L VSV WA TA D WET YN H S N N TQ VR SAADR AS. REV TR EC KT D .SI EYQEPV..VK A FSeu S L S L LI D I V L T VL V PFV ML Y D L V L NR AR L G L VSV WA TA D RKE YN H S N N TQ VE SRRDE GS. RQV DE DV KA D .SI RYEEPA..VK A FPsc S L S L LM I I I T VL V PFI VV Y D V L NR AR L G L SV WAT A RD NAD YS V S N N TR IE SNDDDFDS. RSV DI SK AC E .KV KYVQPM..IDT A FSMOE7 S L S L MLV E L V I SDIL V P I MV F D GL L I NR VR L G V ASVG H N GLP EK Y G L A RR SE AAAG EE. HRF AG AM AE A LPG TVPDPD..SR E..Sly S L S L MLV E L V I SDIL V P I MV F D GL L I NR VR L G V ASVG H N GLP EK Y G L A RR SE AAAG EE. HRF AG AM AE A LPG TVPDPD..SR E..SwMppP S L S L LLV E I V I SDIL V P I LI F D GL L I NR VR L G V SVG F D GLP EK Y G L A RE SR AADG AD. HAF LH SV RA A VEG SFPDPE..SRS E.. TT . TT TT ..SwMppP .. . ........ Plu4 320 330 340 350 360 370 380 Plu4 LP F W R L I L I V GR I V MK F I L AVD SATGYDDVSFAAR KEHD A F Y NSKSQHTQF.. S PDAE YEG GK KE TR.ILEK........S-31 LP F W R L L V L V V GR V V MK F L LMSTAGTG DDRT VRE RGHG G G F DNPNAADSTRN S PDGE ERG A AEYFDK.AVRR........Psy4 LP F W R L L L L I GR V I LK F M LL LDCRLTG TDIEFCKA ATSG G N H ESSAHCSNHFN S KGSD KRA I SGF NTWSPSAAGTSLGHHInd4 LP F W R L I L L L V I G G I A AR F L ALR DHPS RSDD VEM AKSQ G DH Y DDHET SHF.. F EERW ATACGS RE LA.RPELIEGAGS..Bfl4 LP F W R L I I V M L V G G I IA AR F AL IN ADPS NSLH LES LEFG G DH H ANPEV ERY.. DPKM RKSCD SRY EL.SNLKMKEEVR..Pti4 LP F W R L I V M L L I G G I IA AR F IL ALR TDPN RSLD VDR QRLG G DH Y HDHGQ ERY.. CPNM AEACN QS MTNRMDSV.......RohP LP F W R L V L V L V V GR G V MA AR F M L VLR DHPE TATD HRL SADG Y Y Y SEPSK DAY.. EPEM ADA A TRQ DR.HGR.........Seu LP F W R L I L L L V V GR G V LA AR F M VLR DHPE RASE QRL AIEG Y Y F SQPGK EAY.. EPRL AEA VRTRE DR.HA..........Psc LP F W R L I V L L V V GK G I LA AR F V AI ALE LPAS NADD QAY LQSD Y Y Y SQPEK QRY.. KPDE EAA Q AQ EN.VHV.........SMOE7 LP F W R L V L V L V A R G L LA AR V AL VLR GIDD TGTQ WDR REHN Y C P H ANPTE DHT.. STDPLAQS R RH KN.R...........Sly LP F W R L V L V L V A R G L LA AR V AL VLR GIDD TGTQ WDR REHN Y C P H ANPAE DHT.. STDPLAQS R RH KN.R...........SwMppP LP F W R L V V L V A R G V IA R V VL VLR AFAGRTGTE WEE QRHH F C Q H AEPSD DHM.. S STEPLEKS Q RT ET.R........... TT .. SwMppP_ ` _ _ _ _ ` _ ` _ ` d _ ` _ ` d _ ` ` _ _ _ _ ` ` d _ ` ` d d ` _ _ ` _ _ _ ` _ ` _ ` _ _ ` _ ` d d _ ` ` _ _ _ ` ` _ ` ` d d ` _ Figure 2.15: Full sequence alignment of representative arginine oxidases. Secondary structural elements are shown above and below for Plu4 and SwMppP, respectively. Residues that were targeted for mutagenesis are highlighted with red arrows. Descriptions of each abbreviated sequence name is given in Table 2.7. 73 2.4 Discussion These results show, for the first time, the X-ray crystal structure of an arginine desaturase. Through these crystal structures, catalytic residues His27 and Asp225 were identified as a catalytic dyad and were demonstrated to have a role in forming 5. While the corresponding His residue has been previously implicated to have a role in forming 3 in the hydroxylases,115,116 the Asp residue, which is also conserved across the arginine oxidases, has not previously been reported for its role in catalysis. The highly conserved nature of the hydroxylase and desaturase active sites shows that the key to distinguishing the two reactivities may be more complex than a mutation of a single active site residue. Additionally, the mutagenesis work described here did not identify any residues that were responsible for distinguishing hydroxylase and desaturase reactivity. Therefore, the key to distinguishing these activities may lie within other aspects of the protein’s structure. There were several residues surrounding the active site of Plu4 that were observed in various conformations in both MppP and RohP structures, but do not appear in different conformations for Plu4. The N-terminal region of the hydroxylases appears more dynamic than in Plu4, demonstrated by lower B-factors in Plu4 and lack of ordering in the hydroxylases without substrate bound (Figure 2.4 and Figure 2.7). Residues Phe119, Asn121, Leu122 appear to change conformations in the different RohP crystal structures, but the corresponding residues do not appear to move in Plu4 (Figure 2.5d, Figure 2.9c and Figure 2.13c). Phe191 rotates 90° in MppP between the internal aldimine and external aldimine but remains in a single conformation in Plu4 (Figure 2.5e, Figure 2.9d and Figure 2.13d). Reduced movement of these residues suggests increased rigidity of the desaturase active sites, which could differentiate the reactions. The conformations of the hydrophobic residues in particular could prevent water from reaching the active site in the desaturases, making the movement and/or position of water in the active site a potential way of distinguishing reactivity. The oxygen reactivity mechanism within the arginine oxidases remains highly elusive as the crystal structures reported here have not shown any direct evidence of a peroxy-intermediate. However, in flavin-dependent enzymes and cofactor-independent enzymes, positively charged species in the active site are thought to contribute to oxygen activation by stabilizing the negatively-charged peroxy-intermediates formed during the reactions.120,121 In Plu4 and perhaps other arginine oxidases, Lys219, Lys227, or the arginine substrate itself could be responsible for 74 providing the positive environment needed to stabilize the formation of a negatively-charged peroxy-intermediate. There is some evidence that such an environment exists in Plu4 by analysis of the electrostatic map of the active site surrounding the quinonoid intermediate (Figure 2.16). Overall, this work helps to decipher the mechanism of arginine oxidases by providing structural insights for the arginine desaturases and identifying catalytic residues for the first time. A thorough knowledge of these enzymes and their mechanism helps to expand the known biocatalytic repertoire by exploring a new type of enzymatic reaction. Should this new reaction become an attractive tool to synthetic chemists, this work will aid future efforts in enzyme engineering towards a useful biocatalyst by providing a more in depth understanding of the natural enzyme. Complementary to the work described here, product analyses of Plu4 and other Ind4 homologs will be described in Chapter 3. Further work to explore the mechanism and other non-structural characteristics of the arginine oxidases will also be analyzed in Chapter 3. Figure 2.16: Electrostatic map of the Plu4 active site pocket surrounding Quinonoid I in the intermediate-bound structure, coloured according to surface potential as indicated in the figure legend with values given in kT/e. The positively charged pocket near the phosphate group is formed by Lys219 and Lys227. Map was created using the APBS Electrostatics Plugin in PyMOL.236 75 Chapter 3: Understanding the evolutionary relationship and bioactivity of arginine desaturases4 3.1 Introduction In Chapter 2, a deeper understanding of the arginine oxidases was accomplished by obtaining the first crystal structure of an oxygen- and PLP-dependent arginine desaturase, Plu4, after attempting to crystallize a number of predicted homologs of the first known arginine desaturase, Ind4.113 Despite being a predicted Ind4 homolog, Plu4 appears from a convergent indolmycin pathway from P. luteoviolacea.68 Plu4’s sequence identity to Ind4 (32%) is also much lower than that of other homologs to Ind4 (>54%) and is more similar to the sequence identity between the hydroxylases and Ind4 (37-41%). Therefore, it is important to further characterize Plu4 and ensure that it behaves similarly to Ind4 through biochemical characterization. Also, despite this new structural information, no active site residues were implicated to distinguish the hydroxylation and desaturation activities in the arginine oxidases, making the mechanism of differentiation still highly elusive. Therefore, other characteristics of these enzymes must be explored to gain a better insight into their mechanism. An aspect of all arginine oxidases that remains to be understood is their oxygen reactivity. In the hydroxylases, the hydroxyl group is derived from water, which makes the role of molecular oxygen elusive as no atoms from the co-substrate are incorporated into the final product for either the hydroxylases or desaturases. The current understanding of arginine oxidase reactivity with oxygen comes from a comparison with flavo-proteins. The nature of oxygen reactivity in the flavo-proteins has been debated and widely investigated, but they are thought to catalyze a single electron transfer to oxygen, forming superoxide and a semi-quinone as a caged radical pair, which can have a variable fate depending on the enzyme.120,121 While superoxide peroxy-intermediates have been proposed as reaction intermediates in the arginine oxidases, their existence and fate during catalysis have not been explored in these enzymes. In this chapter, the sequence similarity relationships amongst the arginine oxidases are analyzed to better understand potential evolutionary origins and be able to predict hydroxylase or desaturase activity in uncharacterized enzymes. This knowledge will be helpful to identify new 4 A version of this chapter, in combination with Chapter 2, has been submitted for publication. Some of the experiments and calculations described in this chapter was completed by collaborators. 76 potential biocatalyst candidates and aid in any future efforts to understand how these reactivities evolved. The biochemical activities of the desaturase homologs used in crystallographic studies are also characterized to confirm their identities as desaturases. Plu4 was investigated further with a more in-depth look at its kinetics, side reactions and oxygen reactivity. Then, armed with the crystallographic and mutagenesis data described in Chapter 2, a unified mechanism for the arginine hydroxylases and desaturases is proposed that accounts for the new information that will be described. 3.2 Materials and methods 3.2.1 General methods, materials and strains Reagents, columns and primers were purchased from the same sources as listed in Chapter 2, except for the use of materials from Hampton Research. Sequencing was carried out as described in Chapter 2. A list of strains and plasmids used in this study is given in Table 3.1. Table 3.1: Bacterial strains and plasmids used in this study. Strain/Vector Description Source Strain E. coli DH5α General cloning host Laboratory stock E. coli BL21 (DE3) Host for protein expression Laboratory stock Plasmid pET22b Vector for protein expression in E. coli Laboratory stock pET28a Vector for protein expression in E. coli Laboratory stock pET22b-ind4 Vector for Ind4 expression, cloning sites NdeI/XhoI Du et al.48 pET22b-pel4 Vector for Pel4 expression, cloning sites NdeI/XhoI Du et al.48 pET28a-swmppP Vector for SwMppP expression, cloning sites NdeI/XhoI This study pET22b-plu4 Vector for Plu4 expression, cloning sites NdeI/XhoI This study pET22b-bfl4 Vector for Bfl4 expression, cloning sites NdeI/XhoI This study pET22b-pehi4 Vector for Pehi4 expression, cloning sites NdeI/XhoI This study pET22b-punc4 Vector for PUNC4 expression, cloning sites NdeI/XhoI This study 77 Strain/Vector Description Source pET22b-bsp4 Vector for Bsp4 expression, cloning sites NdeI/XhoI This study pET22b-pti4 Vector for Pti4 expression, cloning sites NdeI/XhoI This study pET22b-f5135 Vector for F5135 expression, cloning sites NdeI/XhoI This study pET22b-mst1 Vector for P_Mst1 expression, cloning sites NdeI/XhoI This study pET22b-m63 Vector for P_M63 expression, cloning sites NdeI/XhoI This study 3.2.2 Network analysis, sequence alignment, phylogenetic trees and homolog identification Sequence similarity networks (SSNs) were made using Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST)201–204 from the top 1000 hits in an Ind4 search of the UniprotKB database with an initial search threshold of 10-30. The threshold was increased to 10-65 and 10-85 to observe separation of nodes with different reactivities. Genomic neighbourhood analysis was done using Enzyme Function Initiative-Genomic Neighbourhood Tool (EFI-GNT).201–204 Networks were analyzed using Cytoscape.205 A sequence alignment was made using Muscle in UGENE.175 Phylogenetic trees were made from the alignment using IQtree206–208 using the top 1000 BLASTp hits from Ind4. LG+F+I+G4 was used as a substitution model and the resulting tree was visualized using Interactive Tree of Life.209 Homologs were identified from a BLASTp search of Ind4 by having sequence similarity above 50% or were from known indolmycin producers. These homologs were cross referenced with the UniprotKB database to determine their location in the SSN. Homologs chosen for characterization had between 50%-95% sequence identity to Ind4 and had less than 96% sequence identity to other chosen homologs. Ten homologs of Ind4 (Table 3.2) fit this criteria and attempts were made to further clone, purify and characterize them. 78 Table 3.2: Enzymes used in this study, including arginine desaturase library and hydroxylase control. Homologs were chosen from a BLASTp search of Ind4 (March 2017). Enzymes with unknown activity were not tested for activity. Homolog Species Activity Accession Number Size (aa) Identity (%) Extinction coefficient (M-1cm-1) Ind4 Streptomyces griseus ATCC 12648 Desaturase AJT38685.1 383 100 43,430 Pel4 Paenibacillus elgii B69 Desaturase WP_139074851.1 382 59 46,410 Plu4 Pseudoalteromonas luteoviolacea strain HI1 Desaturase WP_039608190.1 381 32 51,340 Bfl4 Bacillus flexus NBRC 15715 Desaturase WP_061786414.1 383 54 45,380 Bsp4 Bacillus sp. 5mfcol3.1 Desaturase WP_090977143.1 384 56 47,330 Pti4 Paenibacillus tianmuensis strain CGMCC 1.8946 Desaturase WP_143006797.1 382 59 47,900 F5135 Streptomyces sp. NRRL F-5135 Desaturase WP_051765546.1 386 95 44,920 Pehi4 Paenibacillus ehimensis A2 Desaturase WP_152547756.1 382 59 71,390 P_Mst1 Paenibacillus tyrfis strain MSt1 Unknown WP_036690068.1 382 59 46,410 P_M63 Paenibacillus elgii strain M63 Unknown KZE74284.1 378 59 40,910 Punc4 Paenibacillus sp. UNC496MF Desaturase SFI40191.1 382 58 49,390 SwMppP Streptomyces wadayamensis strain A23 Hydroxylase KDR62041.1 376 37 43,430 3.2.3 Cloning Genes F5135, Pehi4, P_Mst1, P_M63, Pti4, Punc4, Bfl4, and Bsp4 were synthesized by Bio Basic Inc. DNA coding for the chosen homologs were synthesized in the pUC57-Kan vector. The homolog genes were sub-cloned into pET22b using NdeI and XhoI to contain a C-terminal histidine tag. SwMppP,114,115 which was used as a hydroxylase control, was obtained through gene synthesis in pUC57-Amp and the swmppP gene was sub-cloned into pET28a using NdeI and XhoI to contain an N-terminal histidine tag. The resulting vectors were transformed into chemically competent E. coli DH5a by heat shocking followed by selection on LB plates 79 containing ampicillin (100 µg/mL) or kanamycin (50 µg/mL). Colonies were screened by colony PCR with T7 primers (Table 3.3). Plasmids from successful colonies were purified using the QIAprep Spin Miniprep Kit from Qiagen and confirmed by sequencing. Successful plasmids were transformed into chemically competent E. coli BL21 (DE3) by heat shocking for protein expression. Expression vectors, containing ind4,48 pel4,113 and plu4 (unpublished) were made previously using pET22b. Table 3.3: Oligonucleotide primers used in this study. Primer Sequence (5′ to 3′) Description T7 TAATACGACTCACTATAGGG T7 promoter for sequencing T7-term GCTAGTTATTGCTCAGCGG T7 terminator for sequencing 3.2.4 Protein purification Bacterial growth, protein expression, cell lysis and purification were done as described in Chapter 2, but the protein was not dialyzed in hydroxylamine to remove all bound PLP and enzymatic products. Instead, fractions collected from the gel filtration column were pooled and directly dialyzed overnight in Buffer D containing 10% glycerol and PLP in five times molar excess of the protein concentration to reconstitute the PLP. The dialyzed samples were concentrated, aliquoted and stored as described in Chapter 2. Enzymes P_Mst1 and P_M63 did not express well enough to produce a sufficient amount of material from the Ni-charged column purification step, so work with these proteins was discontinued. 3.2.5 In vitro characterization Each homolog was assayed by ESI-MS, as described in Chapter 2 for variant characterization. For analysis of products derivatized with o-phenylenediamine (OPD), the 100 µL overnight reaction was treated with 100 µL of water and 100 µL of an OPD solution (100 mM OPD in 2 M HCl) for 20 min, at 80 °C, unless otherwise specified. Reactions containing no enzyme, no L-arginine, or boiled enzyme were used as negative controls. The derivatization time was optimized using the SwMppP reaction to reduce the dehydration side reaction and increase detection of 3. The derivatized reactions were centrifuged to remove denatured protein and 20 µL of the supernatants were analyzed on a 6120 Quadrupole LC-MS system (Agilent) using 80 a Luna C18(2), 5 μm, 4.6 mm ID × 250 mm column (Phenomenex). Elution was done using a mixed mobile-phase linear gradient of water and acetonitrile ((v/v): 95:5 to 50:50, 0 to 15 minutes; 0:100, 15 to 22 minutes; 95:5 22 to 30 minutes), each containing 0.1% (v/v) formic acid. Detection was done in positive ion mode. The OPD-derivatized reaction products were quantified by high-performance liquid chromatography (HPLC) using a 1260 HPLC apparatus (Agilent) system using the same column and elution conditions listed above for the LC-MS system. The hydroxylation side-product was further confirmed by running the overnight reaction in 50% H218O. Derivatization was done over 2 min at 80 °C to reduce dehydration of derivatized 4-hydroxy-2-ketoarginine and allow for detection of the 18O labelled product. Plu4 kinetic assays were performed by monitoring the consumption of O2 using a Clark-type polarographic electrode OXYG1 (Hansatech).113,116 The electrode was calibrated daily according to manufacturer’s instructions using air-saturated water and sodium hydrosulfite. The standard reaction was performed in 1 mL of air-saturated 40 mM TAPS buffer (I = 0.1 M, pH 8.5) at 25°C; buffer and L-arginine mixtures were pre-equilibrated in the reaction chamber prior to reaction initiation by the addition of 2.5 µM Plu4. Reaction rates were measured from the slope at maximum velocity, averaged over at least one minute, and corrected by subtracting the average background oxygen consumption rates prior to reaction initiation. The effect of pH on the rate of Plu4-catalyzed reaction was determined by using air-saturated 20 mM buffers of HEPES (pH 7.5), Tris-HCl (pH 8.0, 8.5 and 9.0), CHES (pH 9.4), and CAPS (pH 10.4). Steady-state kinetic parameters of Plu4 for L-arginine were measured in air-saturated buffer with varying concentrations of L-arginine (5 – 120 µM). The steady-state kinetic parameters for O2 were evaluated using 150 µM of 1 and initial concentrations of O2 from 14 to 256 µM. The initial O2 concentrations were achieved by equilibrating the reaction mixture with humidified mixtures of O2 and N2 gasses. Final O2 levels were normalized to the ambient O2 level prior to the adjustment. The electrode was equilibrated with air-saturated buffer between runs. Steady-state kinetic parameters were evaluated by fitting the Michaelis-Menten equation to the data using the least-squares fitting of LEONORA.210 Plu4 was assayed in the presence of cytochrome c to detect the presence of superoxide. Detection of superoxide was done by including 0.7 mg/mL cytochrome c in a Plu4 reaction containing 5 µM Plu4, 1 mM L-arginine, 25 µg/mL catalase in Tris-HCl pH 8.5. Reactions were initiated by addition of L-arginine, after which UV signals were measured immediately, after 2 81 min and after 4 min. The absorbance signal was obtained on a Cary 100 UV-Vis spectrophotometer. Negative controls, which excluded either Plu4 or L-arginine, were compared to the samples containing both the enzyme and L-arginine. Rates of cytochrome c reduction were measured from the absorbance signal at 550 nm. The linear trendline was determined from the maximal velocity measured over one minute. Rates were measured in triplicate. The rate of cytochrome c reduction in the presence of 1000U/mL superoxide dismutase (SOD) was also determined in triplicate to determine if SOD inhibits cytochrome c reduction. 3.2.6 Electron paramagnetic resonance (EPR) Reactions were done by combining 0.5 mM of compound 1, 10 µM Plu4 and 1 mM cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) in 50 mM Tris pH 8.5 buffer. The buffer was mixed with CHELEX (5 g/ 100 mL) for 7 hours and then mixed with 4 mg DTPA (100 µM) to remove metals that could interfere with CMH. Controls were done using either CMH only, CMH and 1 only, or CMH, 1 and boiled Plu4. The full reaction with active Plu4 and the boiled Plu4 control reaction were done in triplicate. Either 25 µg/mL of catalase or 0.5 mM hydrogen peroxide were used in the assays to test the potential effect of the hydrogen peroxide product on the CMH signal. Electron paramagnetic resonance (EPR) spectra were collected at room temperature at X-band (9.80-9.84 GHz) using a Bruker EMXplus spectrometer, a PremiumX microwave bridge, and a HS resonator. Samples were prepared by adding an aliquot (~300 μL) of either a control or reaction solution into a quartz flat cell ensuring sufficiently high resonant Q-factor for the measurement of aqueous samples. General experimental parameters: frequency = 9.81 GHz, microwave power = 2.0 mW, time constant = 10.24 ms, modulation amplitude = 3.0 G, where each spectrum is an average of five 15-second scans. All spectral centres were calibrated to a frequency of 9.81 GHz. 3.3 Results 3.3.1 Sequence similarity networks In order to better understand the relationship between the two types of arginine oxidases, SSNs were made to help map out potential evolutionary relationships within the arginine oxidases. Also, by assigning the unknown enzymes to specific functions, the sequence 82 similarities and differences could be used to build a hypothesis for the enzymes’ reactivity. Using Ind4 as a query, an initial SSN with a threshold of 10-30 was made from the top thousand sequences and showed that the known arginine oxidases are highly isolated from the other top hits, which were uncharacterized aminotransferases (Figure 3.1). Another SSN was made only using the 296 sequences that clustered together with the known arginine oxidases at a threshold of 10-65, revealing three distinct groups labelled Groups I-III (Figure 3.1). Next, the genomic context of each of these nodes was analyzed to assign probable function to uncharacterized nodes based on neighbouring genes. Over 14 different gene organizations were observed (Appendix Figure A-1, Appendix Figure A-2, Appendix Figure A-3 and Appendix Figure A-4), five of which had known functions in the production of four natural products: enduracididine, azomycin, mildiomycin and indolmycin (both the P. luteoviolacea and S. griseus ATCC 12648 gene organizations were observed). Group II had a large portion of its enzymes (~57%) contained in enduracididine biosynthetic gene clusters,164,166 but at least two other uncharacterized non-enduracididine gene clusters were identified (Appendix Figure A-2). Since the majority of the enzymes in this group are in enduracididine gene clusters, this group is also likely to consist of mostly hydroxylases. Group III contains Plu4 from the P. luteoviolacea indolmycin gene cluster (Appendix Figure A-4), but the incidence of the indolmycin gene cluster within this group is fairly limited. However, other enzymes in Group III are co-localized with plu5-like genes and sometimes ind3-like genes (Appendix Figure A-4). Currently, colocalization with an ind5/plu5-like gene is an invariable quality of the arginine desaturases. Although it is theoretically possible for an arginine desaturase to exist independent of a colocalized ind5/plu5-like gene, or for an arginine hydroxylase to have an ind5/plu5-like counterpart in its gene cluster, further characterization of the relationship between arginine desaturases and their ind5/plu5-like counterparts would be needed to disprove this colocalization pattern as an essential characteristic of arginine desaturase activity in vivo. Based on the current knowledge, these colocalized genes suggest that many members of Group III could function as arginine desaturases. Despite the seemingly consistent clustering of activities in Groups II and III, Group I consisted of diverse gene clusters and previously characterized hydroxylases and desaturases, making the organization of this group non-isofunctional. Therefore, Group I was further analyzed at a 10-85 threshold, revealing three isofunctional sub-groups: Ia, Ib, and Ic (Figure 83 3.1). Group Ia contained the characterized enzyme, RohP,116 and the four-gene azomycin gene cluster173 was a highly conserved gene organization from this sub-group (Appendix Figure A-1a). Additionally, one enzyme in Group Ia was found in the mildiomycin biosynthetic gene cluster (Appendix Figure A-1b), but the enzyme has not been characterized.211 Since mildiomycin contains a 4-hydroxy-2-ketoarginine side chain, this enzyme is also likely a hydroxylase. Therefore, Group Ia consists of known and predicted arginine hydroxylases. Group Ib consisted of only two nodes contained in previously known gene organizations, both of which are from S. griseus ATCC 12648-like indolmycin gene clusters48,113 (Appendix Figure A-3a). One other enzyme from Group Ib, Pel4, is from an uncharacterized gene cluster, but has also been shown to have desaturase activity.113 The gene organization surrounding the pel4 gene was similar to several other nodes in Group Ib (Appendix Figure A-3b). The remaining nodes were found in an unknown gene cluster from Bacillus (Appendix Figure A-3c), making it unclear if they were desaturases. However, the Bacillus nodes were also colocalized with an ind5-like gene, suggesting that they are likely to function as arginine desaturases. Therefore, Group Ib consists of nodes that are predicted to be arginine desaturases. The function of Group Ic nodes and their associated gene clusters remain unknown. Through analysis of these SSNs, it was expected that the hydroxylase nodes would cluster together with higher sequence similarity than with any desaturase nodes. The same pattern was expected for the desaturases. However, the Ind4-like desaturases from Group Ib and the RohP-like hydroxylases from Group Ia displayed the strongest sequence similarity relationship amongst all the groups. (Figure 3.1). This sequence similarity patterns suggests that either the hydroxylases or desaturases emerged on at least two different occasions during evolution, which is also mirrored by analysis of the phylogenetic trees (Figure 3.1 and Figure 3.2). This feature could make elucidating the mechanism of differentiation challenging since there could be more than one evolutionary pathway that each group took to reach the same reactivity. 84 1 x 10-301 x 10-65Shape: FunctionHydroxylaseDesaturaseUncharacterizedColour: Gene ClusterEnduracididineAzomycinMildiomycinIndolmycinInd3 and Ind5/Plu5Ind5/Plu5, proximalInd5/Plu5, distalUnknownIIIIII1 x 10-85 IbIcIaPlu4RohPInd4Pel4MppPFigure 3.1: Sequence similarity network analysis for the arginine oxidases. Each node represents a different enzyme from the UniProtKB database and edges represent sequence similarity relationships. Node shapes represent the characterized function of the enzymes, while node colours indicate information about surrounding genes in the genome. Previously characterized nodes and newly characterized Plu4 are labelled. Dashed boxes indicate nodes used for smaller networks at a higher threshold cut-off. These smaller networks at thresholds of 1x10-65 and 1x10-85 are shown as inset boxes. Each predicted reactivity group is labelled as I-III. 85 Group IaGroup IbGroup IcGroup IIGroup IIIa                                                                                                                                                                                                                                                !                  "                                                                                            ##                                                            $"                                                                                         $ %                  "                                         "                  !                                                                             !               "                                     #                            &                                      '                                                                                                                                                    !     %                               $                                                                %                                 *RohP*Ind4*Pel4*Plu4*MppPGroup IaGroup IbGroup IcGroup IIGroup IIIbFigure 3.2: Phylogenetic trees of the arginine oxidase family of enzymes. Trees were created from the top 1000 sequences obtained from a BLASTp search of Ind4 (January 21st, 2019) and are shown as either (a) unrooted, or (b) circular and rooted from distantly related enzyme hits. The distantly related hits are collapsed and shown as a grey triangle for simplicity in both trees. Clades are labelled with their corresponding SSN group and known arginine oxidases are identified with an asterisk and labelled. Uncoloured clades could not be matched to an SSN group for lack of appearance in the UniprotKB database. 86 3.3.2 In vitro desaturase characterization 3.3.2.1 Product analysis and kinetics5 When crystals could not be obtained for Ind4, attempts were made to crystallize one of seven other homologs that were identified, cloned and purified (Chapter 2). In order to confirm these enzymes as Ind4 homologs, an LC-MS based assay was used to analyze the products of each enzyme, with a particular focus on the crystallized homolog, Plu4. Each purified homolog performs an identical reaction to Ind4, producing 174 m/z and 172 m/z signals in the presence of catalase and 1 (Figure 3.3 and Figure 3.4a), corresponding to expected [M+H]+ ions for 4 and 5, respectively (Figure 2.1). During shorter reaction times, it was also shown in Plu4 that this reaction likely traverses through 2 (Figure 2.1) because the corresponding [M+H]+ 171 m/z signal was observed (Figure 3.5). In steady-state kinetic analyses using an oxygraph, Plu4 was most active at a pH of 8.5-9.0 (Figure 3.6). When assayed using TAPS buffer (I = 0.1; pH 8.5) at 25 °C, Plu4 had KM and kcat values of 12 ± 1 µM and 12.9 ± 0.3 min-1, respectively, for 1 and KM and kcat values of 15 ± 1 µM and 14.9 ± 0.3 min-1, respectively, for O2 (Figure 3.4b and c). Altogether, these data support that the identified homologs, including Plu4, are members of the arginine desaturases consistent with their predicted functions. 5 E.K. completed steady-state kinetic analyses and determined kinetic parameters. 87 Pehi4m/z160 170 180 190020406080100162.1170.1 171.1172.1174.1175.1176.1190.0Intensity (%)Max: 7993020406080100171.1172.1174.1175.1176.0 190.1Bfl4m/z160 170 180 190Intensity (%)Max: 10640m/z160 170 180 190020406080100162.1170.1171.1172.1173.1174.1175.1 190.1Pti4Intensity (%)Max: 9132m/z160 170 180 190020406080100173.1170.1 171.1172.1174.1190.1F5135Intensity (%)Max: 12468m/z160 170 180 190020406080100173.1175.1171.1190.1172.1174.1Ind4Intensity (%)Max: 13932020406080100m/z160 170 180 190171.1170.0172.1173.0174.1190.1Punc4Intensity (%)Max: 14696Bsp4m/z160 170 180 190020406080100172.2174.2175.3190.2Intensity (%)Max: 25303m/z160 170 180 190020406080100 172.1174.1175.1190.1Pel4Intensity (%)Max: 16291NH2H2N NH OOO5[M+H]+ 172 m/z4NH2H2N NH OOO[M+H]+ 174 m/zNH2H2N NH OOOOH3[M+H]+ 190 m/zFigure 3.3: LC-MS assay of known and newly discovered arginine desaturases from SSN Group III. Peak heights are not quantitative because ionization efficiencies will differ between each product. The structures of compounds 3, 4, and 5 are given to the left with their corresponding [M+H]+ ions. 88 024681012140 30 60 90 120Rate (µM O2/min)[L-Arginine] (µM)02468101214160 50 100 150 200 250Rate (µM O2/min)[O2] (µM)cKM = 15 ± 1 µMkcat = 14.9 ± 0.3 min-1KM = 12 ± 1 µMkcat = 12.9 ± 0.3 min-1bam/z160 170 180 190020406080100187.9166.2194.1192.1170.1173.1175.0 190.1174.1172.0Intensity (%)Max: 1322910 15 20SwMppPPlu4Plu4Plu4SwMppP262 m/z246 m/z244 m/zRetention Time (min)NNOHOHNHH2NNH[M+H]+ 262 m/zNNOHNHH2NNH[M+H]+ 246 m/zNNOHNHH2NNH[M+H]+ 244 m/zd e f10 15 20Plu4Ind4Bsp4Punc4Bfl4SwMppP262 m/zRetention Time (min)Figure 3.4: Plu4 reaction products and kinetics. (a) LC-MS analysis of Plu4 reaction with 1. (b and c) Steady-state kinetic analyses of the Plu4-catalyzed reaction, with the lines representing fits of the Michaelis-Menten equation to the data: dependence of initial velocity on (b) 1 and (c) O2. Concentrations were measured by the rate of oxygen consumption. (d) Structures of OPD-derivatized products and their corresponding [M+H]+ signals. (e) Comparison of Plu4 and SwMppP products as extracted ion chromatograms from LC-MS after 20 min OPD-incubations. (f) Comparison of OPD-derivatized products from several desaturases and SwMppP. Traces are extracted ion chromatograms at 262 m/z from LC-MS. Peaks corresponding to 3 are highlighted in green, while peaks corresponding to 4 and 5 are highlighted in blue in (e) and (f). 89 m/z160 170 180 190020406080100Max: 15043171.1172.1174.1175.1190.0Intensity (%)Figure 3.5: Plu4 reaction products measured by LC-MS showing didehydroarginine (based on production of an [M+H]+ ion at m/z 171) when the reaction was run for 2.5 h instead of the typical 16 h. All other reaction conditions remained the same as the typical 16 h reaction. 01020304050607080901007 7.5 8 8.5 9 9.5 10 10.5Rate (µM O2/min)pHFigure 3.6: Measurement of Plu4 pH optimum. Reaction rate of Plu4 from the rate of oxygen consumption measured by oxygraph in pH 7.5 (HEPES), 8 (Tris), 8.5 (Tris), 9.0 (Tris), 9.5 (CHES), and 10.4 (CAPS). Each data point represents the average of three measurements and error bars represent the standard deviation. 90 3.3.2.2 Side-product analysis While characterizing Plu4, there was a minor peak at 190 m/z, which matches the mass of 3 (Figure 3.4a). This peak appears consistently in the LC-MS traces for all of the desaturases that were tested, including Ind4 (Figure 3.3). Therefore, attempts were made to determine if this mass corresponded to a minor hydroxylated side-product made by the desaturases, which could help elucidate the evolutionary history and provide key mechanistic details for the arginine oxidases. To determine the identity of this product, OPD was used to derivatize the products of the Plu4 reaction and separate them by LC-MS (Figure 3.4d). Analysis of the derivatized products revealed a small peak with the same [M+H]+ signal of 262 m/z and retention time as derivatized 3 from SwMppP (Figure 3.4e). The presence of this peak was also confirmed in reactions with other desaturases, including Ind4, under the same reaction conditions (Figure 3.4). The new peak observed in the Plu4 reaction does not appear when the enzyme or 1 are omitted, or when the enzyme has been boiled (Figure 3.7a), suggesting that it arises from an enzymatic process involving 1. To further validate that it arises from an enzymatic reaction, the signal from non-derivatized products did not increase after the reaction mixture was separated from the enzyme (Figure 3.7b). Additionally, similar to the formation of 3 in the MppP and RohP reactions,115,116 by assaying the enzyme in 50% H218O it was demonstrated that this side-product incorporated water during formation (Figure 3.8). SwMppP reactions were also analyzed to see if a product consistent with OPD-derivatized 5 could be detected. The SwMppP reaction produced a signal with an [M+H]+ signal of 244 m/z, consistent with the OPD-derivatized 5; however, the appearance of the 244 m/z peak is concurrent with the disappearance of the 262 m/z signal during longer derivatization times (Figure 3.9). Therefore, it is likely that the 244 m/z peak from SwMppP comes from a non-enzymatic dehydration of OPD-derivatized 3 during the derivatization reaction. Using a shorter incubation time of 3 min, the desaturase side-product was quantified by HPLC and compared to the production of 3 from SwMppP. The amount of derivatized 3 was ~35-fold lower than the peak corresponding to derivatized 4 and 5 in Plu4 reactions (Figure 3.10). By contrast in SwMppP, the production of derivatized 3 is ~1.7-fold less than derivatized 4 and 5, suggesting a bona fide difference in reaction outcome for each enzyme, consistent with their biosynthetic roles. 91 0200400600800100012000 10 20 30 40 50Peak Area of TIC Intensity (thousands)Incubation Time (hours)171 m/z172 m/z174 m/z190 m/z10 15 20Boiled Plu4 + ArgArgPlu4Plu4 + ArgRetention Time (min)262 m/za bFigure 3.7: Desaturase side-product controls. (a) Negative controls for Plu4 reaction. Traces are extracted ion chromatograms at 262 m/z from LC-MS for reactions derivatized with OPD. (b) Analysis for non-enzymatic production of hydroxylated side-product. Plu4 reaction products analyzed for various amounts of time by LC-MS after separation from enzyme by filtration. Reactions proceeded for 2.5 h before the enzyme was filtered. Catalase was added to the filtrate to prevent decarboxylation of the a-keto acid products. Each data point represents the average of two replicates and error bars represent the standard deviation. m/z160 170 180 190020406080100166.1162.0171.1192.1194.0173.1177.1175.1190.1176.1172.1174.1Intensity (%)a10 15 20 25Plu4 262 m/zMppP 262 m/zMppP 264 m/zPlu4 264 m/zRetention Time (min)bMax: 9734Figure 3.8: Plu4 assay with 50% H218O. (a) LC-MS assay with small peaks at 192 m/z and 194 m/z. (b) Comparison of OPD-derivatized products from Plu4 and SwMppP assayed in 50% H218O by LC-MS analysis. Derivatization reaction was done at 80 °C for 3 min to minimize a dehydration side reaction. 92 05001000150020002500300035000 10 20 30 40Peak Area of TIC Intensity (thousands)Incubation time (min)244 m/z246 m/z262 m/zNNOHOHNHH2NNH[M+H]+ 262 m/zNNOHNHH2NNH[M+H]+ 244 m/zH+ΔNNOHNHH2NNH[M+H]+ 244 m/zH+Δacb10 15 20244 m/z246 m/z262 m/zRetention Time (min)dm/z160 170 180 190020406080100Max: 25444174.1175.1190.1Intensity (%)Figure 3.9: Optimization of OPD derivatization conditions using SwMppP. (a) Two possible pathways for forming a 244 m/z product from hydroxylated product under the OPD derivatization conditions. (b) LC-MS analysis OPD-derivatized SwMppP reaction products after a 20 min derivatization. Each trace represents an extracted ion chromatogram. (c) OPD-derivatized products from SwMppP at various incubation times at 80°C analyzed by LC-MS. (d) LC-MS control showing no products corresponding to dehydro-2-ketoarginine ([M+H]+ 172 m/z) before derivatization. 93 3.3.2.3 Demonstrating the existence of superoxide6 To better understand the role of oxygen in the arginine oxidase reactions, EPR and a spin trap, CMH, was used to look for the presence of superoxide during the course of the reaction.123,212 To do so, 0.5 mM of 1 and 1 mM of Plu4 were combined in the presence of 1 mM CMH to detect superoxide during the reaction. A four-fold enhanced signal for CMH-trapped superoxide was observed when both Plu4 and 1 were present (Figure 3.11 and Figure 3.12a). When catalase was added to the reaction, the observed signal dropped to a two-fold increase and 6 G.A.M. analyzed the EPR samples and helped with data processing. 0100020003000400050006000Plu4 SwMppPPeak Area (330 nm)4-hydroxy-2-ketoarginine4,5-dehydro-2-ketoarginine/2-ketoarginine35X1.7XFigure 3.10: Quantification of hydroxylated product comparing Plu4 and SwMppP. Analysis was done on OPD-derivatized products by integrating peak areas from HPLC at 330 nm. 4,5-dehydro-2-ketoarginine and 2-ketoarginine are combined into a single bar because they have the same retention time. Each bar represents the average of two replicates and error bars represent the standard deviations. The fold-difference between the hydroxylated product and the other products is indicated. Derivatization reaction was done at 80°C for 3 min to minimize a dehydration side reaction. 94 the signal increased slightly when only Plu4 and hydrogen peroxide were included (Figure 3.12b). These controls indicate that the hydrogen peroxide produced during catalysis is able to react with CMH to some degree, but the remaining two-fold signal from the catalase-containing reaction indicates that superoxide is produced from the Plu4 reaction. To further confirm the presence of superoxide as an intermediate in the reaction, Plu4 was assayed in the presence of cytochrome c, which uses superoxide to reduce Fe3+ to Fe2+, producing a UV-Vis signal at 550 nm (Figure 3.13). Superoxide dismutase (SOD) was also included in the assay to observe if Fe3+ reduction by cytochrome c was inhibited. The rate of reduction for the SOD-free reaction was 21.1 ± 0.5 µM min-1, while the rate of reduction was 15.4 ± 0.5 µM min-1 when SOD was included in the assay. These results demonstrate that superoxide is present as an intermediate in the reaction because the inclusion of SOD resulted in a decreased rate as SOD lowered the amount of superoxide available to reduce the Fe3+. Interestingly, the SOD-free rate of Fe3+ reduction was similar to reaction rates measured from the oxygraph experiments. Magnetic Field Strength (G)viviiiiii3465 3475 3485 3495 3505 3515 3525NOHO OO2 NOO OH+H2O2EPR silent EPR activeFigure 3.11: EPR measurements demonstrating production of superoxide. EPR spectra is from CMH spin-trap radical adduct, shown above the spectra. Traces show (i) CMH, (ii) CMH and L-Arg, (iii) CMH and Plu4, (iv) CMH, L-Arg and boiled Plu4, (v) CMH, L-Arg and Plu4. 95 3460 3470 3480 3490 3500 3510 3520 3530Magnetic Field Strength (G)Plu4-1Plu4-2Plu4-3Plu4 (boiled)-3Plu4 (boiled)-2Plu4 (boiled)-13460 3470 3480 3490 3500 3510 3520 3530Magnetic Field Strength (G)Plu4Plu4, ArgPlu4, Arg, catalasePlu4, H2O2a bFigure 3.12: EPR spectra of CMH-trapped superoxide during control reactions of ʟ-Arg with Plu4. (a) Comparison of reactions with active Plu4 and boiled Plu4 done in triplicate to demonstrate reproducibility. (b) Effect of hydrogen peroxide on CMH signal using catalase to consume the hydrogen peroxide by-product of the Plu4 reaction with arginine. A Plu4 sample without arginine was also spiked with hydrogen peroxide. 0.000.200.400.600.801.001.20450 470 490 510 530 550 570 590 610Absorbance (A.U)Wavelength (nm)Plu4 and arginineno Plu4no arginineFigure 3.13: Absorbance spectra of Plu4 reaction assayed in the presence of cytochrome c. UV-Vis absorbance at 550 nm is used to detect reduction of Fe3+ to Fe2+ by superoxide. Cytochrome c reduction is not present when Plu4 or arginine are eliminated from the assay. 96 3.3.3 Density functional theory calculations7 To further investigate the role of superoxide, the likelihood of possible mechanisms was explored though DFT calculations. To summarize this investigation, a superoxide rebound mechanism was shown to be more favourable than a hydrogen abstraction mechanism for both oxygen reaction steps. Energies calculated for each possible peroxy-intermediate suggests that oxygen reacts at the C4′ position of PLP for both Quinonoid I and II. Finally, a comparison of the hydroxylation and deprotonation steps shows that the hydroxylation pathway was enthalpically favoured, which supports the fact that Plu4 has a minor hydroxylation activity despite being a desaturase. The enthalpic favourability of hydroxylation over deprotonation further validates that the key distinction between hydroxylation and double bond formation could be the position of water in the active site of the enzymes during catalysis, a proposal that was initially introduced in Chapter 2, Section 2.4. 3.4 Discussion The oxygen reactivity demonstrated by the arginine oxidases shows the catalytic potential of PLP-dependent enzymes in their ability to catalyze challenging oxidation reactions without the use of a metal cofactor. Building upon the structural and mutagenesis work described in Chapter 2, the work described here dives further into the biochemical and evolutionary characteristics of these enzymes with a particular focus on the newly characterized Plu4 desaturase from P. luteoviolacea. A look at the SSNs for arginine oxidases revealed that there was a stronger evolutionary relationship between the Group Ia hydroxylases and Group Ib desaturases than between the Group Ia hydroxylases and the Group II hydroxylases or between Group Ib desaturases and Group III desaturases. This result demonstrates that there are at least four distinct phylogenetic groups of arginine oxidases (two hydroxylases and two desaturases) that have now been characterized and that either the hydroxylase or desaturase activity emerged separately on more than one occasion, indicating a complex evolutionary history. Furthermore, consistent appearance of a minor hydroxylated side-product in all characterized desaturases indicates that the hydroxylase and desaturase activities are highly related and provide evidence for promiscuous enzyme evolution.213 The minor appearance of 3 from the desaturases could 7 K.C.H. completed DFT calculations, described in further detail in publication. 97 have either served as a starting point for the evolution of hydroxylation reactions from a desaturase ancestor, or it could represent a vestigial feature of a hydroxylating ancestor. Examples of such a complex evolutionary history and promiscuous enzyme evolution have also been seen for other protein families. The o-succinylbenzoate synthase/N-succinylamino acid racemase (OSBS/NSAR) family is a similarly complex example of non-isofunctional phylogenetic clustering, in which a group of OSBSs share a closer evolutionary relationship with the NSARs (>40% sequence identity) than they do with other OSBSs (Figure 3.14a).214–216 Additionally, independent evolution of a group of NSARs was also discovered, having a closer evolutionary relationship with ʟ-Ala-D/ʟ-Glu epimerases (AEEs) than with other NSARs.215 Another example comes from the 6-pyruvoyl-tetrahydropterin synthases (PTPSs),217,218 which demonstrated that some of the six reactivities observed from these enzymes did not cluster isofunctionally in an SSN, suggesting that each function may have evolved on more than one occasion (Figure 3.14b).217 This complex sequence similarity pattern results in the PTPSs being commonly misannotated without any genomic context.217,219 Since the arginine oxidase reactivities are most commonly identified based on similar genomic neighbourhoods, automated identification of the arginine desaturases and hydroxylases could also result in misannotations. Additionally, some PTPSs promiscuously catalyze two reactions, pointing towards a potential starting point for evolution of other reactions.218,220 Promiscuous enzyme evolution is thought to contribute to the appearance of new reactions by acting as a starting point to evolve a side reaction into an enzyme’s primary function and contributes to the chemical diversity of natural products.213,221,222 Brown and Babbitt state that reaction promiscuity and independent appearances of the same reactivity within a superfamily’s phylogeny often complicates attempts to understand enzyme evolution and predict reactivity from sequence.223 The EPR experiments and cytochrome c assays described here show for the first time that superoxide is an intermediate to reaction within the arginine oxidases. Although only demonstrated for an arginine desaturase, the high similarity with the hydroxylases implies that the hydroxylase reactions also likely include superoxide as an intermediate. A superoxide intermediate has been demonstrated previously for the paracatalytic oxidative deamination reaction of DDC.125 Additionally, flavin-dependent enzymes and cofactor-independent enzymes also produce superoxide through a single electron transfer to oxygen and will serve as good models for further studies of oxygen activation in the arginine oxidases.120,121,123,128 DFT 98 calculations showed that superoxide rebound at the C4′-position of PLP is the most favourable, resulting in a peroxy-intermediate. This result is unprecedented in the arginine oxidases as previous mechanisms have proposed peroxy-adduct formation at the Ca or direct deprotonation of arginine protons by superoxide.113–115 However, formation of the peroxy-adduct at the C4′-position of PLP can be rationalized by the arginine oxidases’ close evolutionary relationship to, and frequent annotation as aspartate aminotransferases, whose mechanisms typically include protonation at the C4′-position of PLP. Alternatively, some of the other oxygen- and PLP-dependent enzymes, such as Cap15119 and CuaB117, show evidence that points towards peroxy-intermediate formation at the Ca position of their substrate, based on the appearance of labelled oxygen atoms being incorporated into their products. These results again can be rationalized by the similarity of Cap15 and CuaB with selenocysteine synthases and AONS, respectively (Table 1.1), which both involve protonation or carbon-carbon bond formation at the Ca position of the substrate. Therefore, it is possible that the oxygen reactivity sites in the oxygen- and PLP-dependent enzymes could be predicted based on knowing the site of new bond-formation in the nearest characterized relatives. The key difference between these desaturases and hydroxylases that aids in the proposal of a new, unified mechanism for the arginine oxidases is the use of water. While the hydroxylases require water to produce 3, water is not a reactant needed to produce 2, making the movement of water a potentially critical factor in differentiating the arginine oxidase reactivity. As discussed in Chapter 2, mutagenesis revealed that there were no single active site residues that differentiated reactivity in the arginine oxidases. Additionally, lower N-terminal B-factors and single conformations of active site hydrophobic residues were observed in the Plu4 crystal structure while the corresponding active site residues in the hydroxylases were observed in multiple conformations and the N-termini had higher B-factors. Therefore, the active sites of the hydroxylases appear to be more dynamic than the desaturases, making it potentially more open to water from outside the active site. Besides the structural differences, the enzymes have highly similar active sites, including the catalytic His/Asp dyad, which supports that water may be the distinguishing factor. A comparison of the final deprotonation and hydroxylation steps by DFT revealed that the hydroxylation step is more enthalpically favourable than deprotonation. This result indicates that the presence of an ordered water in the active site of a desaturase will produce 3 as a side-99 product. These calculations also support that the hydroxylation mechanism does not go through a fully oxidized intermediate with deprotonation at C5 because the hydroxylation step would likely precede deprotonation, since it is more favourable. Therefore, the distinguishing feature between the arginine hydroxylases and desaturases may be an inability to preorganize water in the Plu4 active site, with His27 deprotonating the C5 position of 1 (Figure 3.15). Alternatively, the active sites of RohP and SwMppP would be able to preorganize water and the active site His would act to deprotonate water instead of C5. This proposal is not unprecedented as water has been discussed previously to play a role in distinguishing promiscuous activities in some enzymes.213,224,225 Overall, the arginine desaturases and hydroxylases show a high degree of similarity despite catalyzing different reactions. A reliance on the preorganization of water is proposed as the reaction differentiation mechanism and accounts for the underlying hydroxylation side reaction in the desaturases. A mechanism for oxygen usage is offered that includes a single electron transfer to oxygen and hydroperoxyl-intermediate formation at the C4′ position of PLP, which suggests that the oxygen reactivity is not reliant on arginine as a substrate and could, theoretically, be engineered into other aminotransferases. Finally, the sequence similarity and phylogenetic analysis reveals that the desaturases and hydroxylases appear to each have two distinct phylogenetic groups, clustered non-isofunctionally, which will be essential to future studies aimed at understanding how the two activities evolved. This new information and proposed mechanism will help guide future efforts in biocatalyst engineering and discovery amongst the arginine oxidases, which will expand the biocatalytic toolbox and its practicality for use by chemists. 100 OCO2HOHHCO2 OSBSH2O OCO2HCO2SHCHC OSBNH2NHOCO2CO2HNH2NHOCO2CO2HAEENHOCO2RHʟ-Ala-ʟ-Glu ʟ-Ala-ᴅ-GluNHOCO2RHN-Succinyl-ʟ-AA N-Succinyl-ᴅ-AANSARHNN NHNH2NOOPPPOHOHPTPS-IHNN NHHNH2NOOHOPTPS-III HNN NHNH2NOOHH2NTP6-carboxytetrahydropterinH2-pterin-CH2OHabFigure 3.14: Reaction schemes for examples of non-isofunctional phylogenetic clustering. (a) o-succinylbenzoate synthase (OSBS), ʟ-Ala-D/ʟ-Glu epimerases (AEE) and N-succinylamino acid racemase (NSAR) reaction schemes are shown. The OSBSs and NSARs do not cluster isofunctionally and some enzymes show functional promiscuity/bifunctionality with both OSBS and NSAR reaction types.214,215 (b) 6-pyruvoyl-tetrahydropterin synthase (PTPS) reaction schemes are shown for groups I and III, in which some PTPS enzymes catalyze both reactions promiscuously. PTPS-I and -III enzymes do not appear to cluster isofunctionally in a sequence similarity network.217 101 releasefrom cofactorNHOH2-O3PONOONHH2NNH2BH HNHOH2-O3PONOONHH2NNH2 HNHOH2-O3PONOONHH2NNH2LysNH2Quinonoid I NHOH2-O3PONOONHH2NNH2BC⍺C⍺C4′C4′O2NHOH2-O3PONOONHH2NNH2electrontransferO2NHOH2-O3PONOONHH2NNH2OHOHBEnzH2O2EnzNHOH2-O3PONOONHH2NNH2Quinonoid IIC⍺C4′NHOH2-O3PONOONHH2NNH2O2O2electrontransferNHOH2-O3PONOONHH2NNH2OHOHNHNHisH2O2NHOH2-O3PONOONHH2NNH2EnzNHOH2-O3PONOONHH2NNH2OH releasefrom cofactorH2O NH3NHOH2-O3PONOONHH2NNH2OHONHNHisH2O2OHHDesaturasesHydroxylasesH2O NH3releasefrom cofactorH2ONH3432 5OONHH2NNH2OH OOONHH2NNH2NHOONHH2NNH2OOONHH2NNH2OC5C4C3C5C4C3C5C4C31Figure 3.15: Proposed mechanism of arginine desaturases and hydroxylases. Relevant atom labels are shown in blue. Products observed via LC-MS [M+H]+ signals are indicated with a box. The catalytic His residue is highlighted in red. 102 Chapter 4: Engineered production of indolmycin derivatives through semi-synthesis8 4.1 Introduction As was reviewed in Chapter 1, antibiotic resistance threats have reignited interest in indolmycin, leading to the discovery of new activities against MRSA,38 Helicobacter pylori32 and Plasmodium falciparum.39 Despite this revived interest, there have only been two attempts to synthesize or derivatize indolmycin since the discovery of these new bioactivities.58,60 These attempts build upon the first indole-substituted derivatives, 5-hydroxy- and 5-methoxy-indolmycin, made biosynthetically from a natural producer, S. griseus ATCC 12648. Both of these derivatives showed mild improvements to bioactivity against S. aureus and E. coli.67 Continuing the early biosynthetic work from Hornemann et al. (1971),65 the Ryan group identified the indolmycin gene cluster and elucidated the biosynthetic pathway, demonstrating that indolmycin (6) is assembled from tryptophan, 1 and methionine in a three-part process (Figure 4.1). In the first part, 1 is oxidized by Ind4 in an oxygen- and PLP-dependent reaction to 2, which is then reduced by Ind5 and its chaperone, Ind6, to 4,5-dehydro-D-arginine (7). In the second part, which occurs parallel to the first, tryptophan (8) is deaminated by a PLP-dependent transaminase, giving indole pyruvate (9). Compound 9 is then methylated by Ind1, an S-adenosyl methionine (SAM)-dependent C-methyltransferase, to 3-methyl-indole pyruvate (10) which is reduced by Ind2, a nicotinamide adenine dinucleotide (NADH)-dependent ketone reductase, to form indolmycenic acid (11). Then, in the third part, compounds 7 and 11 are coupled in an ATP-dependent fashion by Ind3 and Ind6, resulting in an oxazolinone-cyclized molecule, N-desmethyl-indolmycin, which is finally N-methylated by Ind7, a SAM-dependent N-methyltransferase to form 6. Here, it is predicted that the indolmycin biosynthetic pathway could be used to make 6 in a heterologous host, such as E. coli. Use of E. coli as a host would allow pathway modifications and improvements to be made more easily than in other hosts, as the genetic manipulation tools available for E. coli are numerous. Based on previously observed acceptance of 5-hydroxy- and 5-methoxy-substituted derivatives,67 it is also predicted that the biosynthetic enzymes may be able to accept a wider variety of indole-substituted derivatives of 8, leading to novel indole- 8 A version of this chapter has been submitted for publication. 103 substituted derivatives of 6. Additionally, in order to expand the scope of analogs that could be made, enzymatic methods to generate indole-substituted derivatives of 8 in vivo from indole derivatives were explored, which are cheaper and more diverse than derivatives of 8. Expansion of accessible derivatives of 6 will benefit the search for new antibiotics as some of these derivatives could show bioactivity against new pathogenic targets or they could help overcome the challenges that caused 6 to be forgotten so many years ago. 4.2 Materials and methods 4.2.1 General methods, materials and strains Reagents were purchased from Sigma-Aldrich, Thermo Fisher Scientific Canada, NEB, Bio-Rad, Bio Basic Inc., Gold Biotechnology, and VWR International. The liquid chromatography column used was obtained from Agilent and the semi-preparative HPLC column was from Phenomex. Primers were purchased from IDT. Sequencing was done through the NAPS Unit in the Sequencing and Bioinformatics Consortium at the University of British Columbia. A list of strains and plasmids is given in Table 4.1. Figure 4.1: Indolmycin gene cluster and biosynthetic pathway from S. griseus ATCC 12648. A dashed arrow indicates a predicted reaction based on observed products by LC-MS analysis. 104 Table 4.1: Strains and plasmids used in this study. Name Description Source Strains E. coli DH5a General cloning host Laboratory stock E. coli BL21 (DE3) Host for protein expression Laboratory stock Streptomyces griseus ATCC 12648 Wild-type indolmycin producer ATCC E. coli I120 E. coli carrying pI0COLA and pI1I2ET; tested for in vivo production of indolmycenic acid This study E. coli I1234670P5 E. coli carrying pP5I0COLA, pI1I2ET, pI3I4CDF and pI6I7ACYC; used for in vivo production of indolmycin This study E. coli I1234670TS E. coli carrying pTSI0COLA, pI1I2ET, pI3I4CDF and pI6I7ACYC; used for in vivo production of IMA and IMA derivatives This study Plasmids pCOLAduet-1 Vector for protein expression in E. coli with two multiple cloning sites Laboratory stock pETduet-1 Vector for protein expression in E. coli with two multiple cloning sites Laboratory stock pCDFduet-1 Vector for protein expression in E. coli with two multiple cloning sites Laboratory stock pACYCduet-1 Vector for protein expression in E. coli with two multiple cloning sites Laboratory stock pET28a-pel5 Template for pel5 amplification Du et al.113 pSTB7 (ATCC 37845) Vector carrying trpS from Salmonella enterica ATCC pI0COLA Vector for cloning pel5 and trpS This study pI1ET Vector for cloning ind2 This study pI3CDF Vector for cloning ind4 This study pI6ACYC Vector for cloning ind7 This study pP5I0COLA Vector for ind0 and pel5 expression This study pI1I2ET Vector for ind1 and ind2 expression This study pI3I4CDF Vector for ind3 and ind4 expression This study pI6I7ACYC Vector for ind6 and ind7 expression This study pTSI0COLA Vector for ind0 and trpS expression This study 4.2.2 Cloning The indolmycin biosynthetic genes (ind0, ind1, ind2, ind3, ind4, ind6 and ind7) were PCR-amplified from S. griseus ATCC 12648 genomic DNA using the primers described in Table 4.2. Genes ind1, ind3 and ind6 were cloned using into the NcoI and HindIII restriction sites of MCS1 of pETduet-1, pCDFduet-1 and pACYCduet-1, respectively, using Gibson Assembly (NEB). Genes ind0, ind2, ind4 and ind7 were cloned using T4 ligase into MCS2 of pCOLAduet-1, pI1ET, pI3CDF and pI6ACYC, respectively, using the NdeI and XhoI restriction sites. The ind5 homolog, pel5 from Paenibacillus elgii B69, was PCR-amplified from a pET28a vector containing pel5 described previously,113 and was cloned using T4 ligase into the NcoI and HindIII restriction sites of MCS1 in pI0COLA. The trpS gene from Salmonella enterica was 105 PCR amplified from pSTB7 and cloned using Gibson Assembly into MCS1 of pI0COLA using the NcoI and HindIII restriction sites, replacing pel5 for the production of 11 and its derivatives. The resulting vectors were transformed into electrocompetent E. coli DH5a by electroporation and selection on LB plates containing either kanamycin (50 µg/mL) for pCOLAduet vectors, ampicillin (100 µg/mL) for pETduet vectors, chloramphenicol (34 µg/mL) for pACYCduet vectors, or spectinomycin (100 µg/mL) for pCDFduet vectors. For co-expression of the genes, the appropriate vectors were co-transformed into electrocompetent E. coli BL21 (DE3) by electroporation and successful transformants were selected for on LB plates containing kanamycin (25 µg/mL), ampicillin (50 µg/mL), chloramphenicol (17 µg/mL), and spectinomycin (50 µg/mL). See Table 4.1 for a description of each plasmid combination made in E. coli. Table 4.2: Primers used in this study. Primer Name Sequence (5′ to 3′) Description Ind0_F ATTAGTTAAGTATAAGAAGGAGATATACATATGATCAAGCTGTCGGGAATCACCC Cloning ind0 into pCOLAduet-1 Ind0_R CGGTTTCTTTACCAGACTCGAGTCAGCCCACTCCGGCGAGTT Cloning ind0 into pCOLAduet-1 NcoI-Pel5-F ACTTTAATAAGGAGATATACCATGGATGAGAATCATTTATCTGGATCAACCTACCTAC Cloning pel5 into pI0COLA HindIII-Pel5-R CTTAAGCATTATGCGGCCGCAAGCTTTCATTCGCTTGCCGGCCTCTTC Cloning pel5 into pI0COLA pCOLA-TrpS-F TAATTTTGTTTAACTTTAATAAGGAGATATACATGACAACACTTCTCAACCCCTACTTTG Cloning trpS into pI0COLA pCOLA-TrpS-R ATTATGCGGCCGCAAGCTTTATGCGCGGCTGGCGGC Cloning trpS into pI0COLA Ind1-3_F AACTTTAAGAAGGAGATATACCATGACCAGGACCGATTTCGCC Cloning ind1 into pETduet-1 Ind1-HindIII-R GCATTATGCGGCCGCAAGCTTCATGAAGCCGGCCC Cloning ind1 into pETduet-1 NdeI-Ind2-F TAGTTAAGTATAAGAAGGAGATATACATATGAAGCTGGACGACAAGAGAATTCTC Cloning ind2 into pI1ET Ind2-XhoI-R GTTTCTTTACCAGACTCGAGTCATAGGCCCTTGATGCGACGGG Cloning ind2 into pI1ET Ind3-F TGTTTAACTTTAATAAGGAGATATACCATGAAGGGTGCGAGACAGGAG Cloning ind3 into pCDFduet-1 Ind3-HindIII-R GCATTATGCGGCCGCAAGCTTTACGGGACCAGGCTGATGATCTGGTTG Cloning ind3 into pCDFduet-1 NdeI-Ind4-F2 GTTAAGTATAAGAAGGAGATATACATATGGAACGGTTCAACAATCTGACG Cloning ind4 into pI3CDF Ind4-XhoI-R2 GTTTCTTTACCAGACTCGAGTCATGAACCGGCGCCCTCG Cloning ind4 into pI3CDF Ind6-F TGTTTAACTTTAATAAGGAGATATACCATGTCGTTCGATAACCAGAACAGG Cloning ind6 into pACYCduet-1 106 Primer Name Sequence (5′ to 3′) Description Ind6-HindIII-R GCATTATGCGGCCGCAAGCTTCAGCCGACTCCGATGTCTCCCAGTC Cloning ind6 into pACYCduet-1 NdeI-Ind7-F GTTAAGTATAAGAAGGAGATATACATATGCACACGGACTGGGAGAC Cloning ind7 into pI6ACYC Ind7-XhoI-R GTTTCTTTACCAGACTCGAGTCAGGCTGTTCCGCGCAC Cloning ind7 into pI6ACYC pET Upstream ATGCGTCCGGCGTAGA Sequencing ind1 in pI1ET pACYCduetUP1 GGATCTCGACGCTCTCCCT Sequencing trpS in pTSI0COLA, pel5 in pP5I0COLA, ind3 in pI3CDF, ind6 in pI6ACYC DuetDOWN1 GATTATGCGGCCGTGTACAA Sequencing trpS in pTSI0COLA, ind1 in pI1ET, ind3 in pI3CDF, ind6 in pI6ACYC DuetUP2 TTGTACACGGCCGCATAATC Sequencing ind0 in pI0COLA, ind2 in pI1I2ET, ind4 in pI3I4CDF, ind7 in pI6I7CYC T7-term GCTAGTTATTGCTCAGCGG T7 terminator for sequencing ind0 in pI0COLA, ind2 in pI1I2ET, ind4 in pI3I4CDF, ind7 in pI6I7ACYC TrpS-seq-F1 GCTCAAACGCTATCGGGATGTTTG Sequencing trpS in pTSI0COLA TrpS-seq-F2 GTACGCGAATCTGGTGTTCAATAACG Sequencing trpS in pTSI0COLA TrpS-seq-R1 CCATATAGATACGGCATTTCAGACCCAG Sequencing trpS in pTSI0COLA TrpS-seq-R2 CGTAGCGTTCCATCAGATTTCCCC Sequencing trpS in pTSI0COLA 4.2.3 Expression and feeding E. coli I1234670P5 and I1234670TS were initially grown in 50 mL of LB media containing kanamycin (25 µg/mL), ampicillin (50 µg/mL), chloramphenicol (17 µg/mL) and spectinomycin (50 µg/mL) for profiling metabolites, analyzing substrate scopes and optimizing the feeding procedure. For larger-scale production of 11 and its derivatives, E. coli I1234670TS was grown in two 500 mL portions of LB media containing the antibiotics mentioned above. Cultures were allowed to grow until 0.4-0.6 OD600 at 37°C with shaking at 200 rpm, after which the temperature and shaking were lowered to 16°C and 150 rpm, respectively, for 30 min prior to 107 addition of IPTG at 0.5 mM final concentration to induce expression of the biosynthetic genes. The cultures were incubated for 16 hours at 16°C and 150 rpm. Then, the temperature was raised to 30°C and the cultures were fed solid indoles or substituted indoles to a final concentration of 0.5 mM in each 500 mL culture once per day for two days. Compounds made by the system were harvested 24 hours after the final indole feeding. Use of trpS for improved acceptance of substituted indoles was tested with 5-fluorindole fed to 1 mM final concentration. Feeding optimization was done with 5-fluoroindole at 0.05 mM to 2 mM final concentration for one, two or three days to determine what conditions produced the most 5-fluoro-indolmycenic acid (5F-11). 4.2.4 Extraction and purification of indolmycenic acid and its derivatives Cell cultures were adjusted to pH 3-4 with HCl and then extracted with two volumes of ethyl acetate, which was gravity filtered and evaporated under low pressure. To analyze the metabolite profile and substrate scope, the resulting residues from small-scale test cultures were dissolved in 1 mL of methanol and analyzed by LC-MS using a 6120 Quadrupole LC-MS system (Agilent) operated in positive ion mode on an Agilent 5 TC18(2) 250 x 4.6 mm column using 85:15% water:ACN to 5:95% water:ACN (v/v) with 0.1% (v/v) formic acid run at 1 mL/min for 20 min. Initial tests for biosynthetic production of 6 (Figure 4.6) were analyzed by the same system above, but with a Phenomenex Luna C18(2), 5 μm, 4.6 mm ID × 250 mm column using 95:5% water:ACN to 0:100% water:ACN (v/v) with 0.1% (v/v) formic acid run at 0.6 mL/min for 20 min. When testing the optimal feeding conditions, the metabolites were analyzed by HPLC using a 1260 HPLC apparatus (Agilent) system using the Agilent 5 TC18(2) 250 x 4.6 mm column with a 75:25% water:ACN to 50:50% water:ACN (v/v) gradient containing 0.1% (v/v) formic acid run at 1 mL/min for 25 min. The 5F-11 peak eluted at 21.8 min and the area of the peak was used to determine the optimal feeding conditions based on the largest peak area. Testing omission of unnecessary indolmycin genes (Figure 4.7), was done with the HPLC system and column mentioned above but with a gradient of 85%:15% water:ACN to 60%:40% water:ACN (v/v) with 0.1% (v/v) formic acid run at 1 mL/min for 25 min. For large-scale growth, the resulting residue was dissolved in 6-7 mL of methanol for purification via semi-preparative HPLC. Semi-preparative HPLC was run with an 6120 Quadrupole LC-MS system (Agilent) using a Luna C18(2), 5 μm, 250 mm × 10 mm column (Phenomenex) using a gradient 108 from 70:30% water:ACN (v/v) with 0.1% (v/v) formic acid to 42:58% water:ACN (v/v) at 3.0 mL/min in 12 min. The collected fraction containing the desired peak for 11, or one of its derivatives, was verified by LC-MS using the conditions described above for analyzing the metabolite profile and substrate scope. Compound 11 and its derivatives were lyophilized and the masses were measured to determine the final amounts obtained. High resolution-mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) were used to confirm the identity of 11 and its derivatives. HR-MS was done on a Waters/Micromass LCT time of flight-mass spectrometer (TOF-MS) with electrospray ionization in positive ion mode. NMR analysis was done in dimethyl sulfoxide (DMSO)-d6 with a Bruker Avance 400dir spectrometer. 11 (Appendix Figure B-1, Appendix Figure B-2, Appendix Figure B-3, Appendix Figure B-4, and Appendix Figure B-5): 1H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 2.4 Hz, 1H), 7.08 – 7.01 (m, 1H), 6.96 (t, J = 7.6 Hz, 1H), 4.19 (d, J = 4.6 Hz, 1H), 3.42 (qd, J = 7.0, 4.4 Hz, 1H), 1.25 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 175.26, 136.11, 126.49, 122.40, 120.73, 118.51, 118.14, 116.85, 111.36, 73.99, 34.02, 15.38. 5F-11 (Appendix Figure B-6 and Appendix Figure B-7): 1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 7.31 (dd, J = 8.8, 4.7 Hz, 1H), 7.26 (dd, J = 10.3, 2.6 Hz, 1H), 7.22 (d, J = 2.5 Hz, 1H), 6.88 (td, J = 9.1, 2.6 Hz, 1H), 4.13 (d, J = 4.7 Hz, 1H), 3.37 – 3.31 (m, 1H), 1.24 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 175.14, 156.52 (d, J = 230.5 Hz), 132.77, 126.69 (d, J = 9.7 Hz), 124.63, 117.15 (d, J = 4.7 Hz), 112.20 (d, J = 9.7 Hz), 108.79 (d, J = 26.2 Hz), 103.35 (d, J = 23.2 Hz), 74.12, 34.09, 15.44. NHOHO OHNHOHO OHF109 6F-11 (Appendix Figure B-8 and Appendix Figure B-9): 1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 7.52 (dd, J = 8.7, 5.5 Hz, 1H), 7.16 – 7.05 (m, 2H), 6.82 (ddd, J = 9.7, 8.7, 2.4 Hz, 1H), 4.15 (d, J = 4.6 Hz, 1H), 3.38 (dt, J = 12.0, 6.9 Hz, 1H), 1.23 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 175.16, 158.68 (d, J = 233.5 Hz), 135.88 (d, J = 12.6 Hz), 123.38, 122.99 (d, J = 3.4 Hz), 119.53 (d, J = 10.3 Hz), 117.13, 106.55 (d, J = 24.4 Hz), 97.20 (d, J = 25.4 Hz), 74.02, 34.01, 15.36. 7F-11 (Appendix Figure B-10, Appendix Figure B-11, Appendix Figure B-12, Appendix Figure B-13 and Appendix Figure B-14): 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.20 (d, J = 2.4 Hz, 1H), 6.98 – 6.83 (m, 2H), 4.17 (d, J = 4.6 Hz, 1H), 3.41 (qd, J = 7.0, 4.4 Hz, 1H), 1.25 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 175.13, 149.31 (d, J = 242.4 Hz), 130.61 (d, J = 6.0 Hz), 123.80 (d, J = 13.0 Hz), 123.66, 118.47 (d, J = 6.1 Hz), 118.07 (d, J = 2.0 Hz), 114.83 (d, J = 3.3 Hz), 105.56 (d, J = 15.9 Hz), 73.89, 34.07, 15.37. 5Cl-11 (Appendix Figure B-15 and Appendix Figure B-16): 1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.04 (dd, J = 8.6, 2.1 Hz, 1H), 4.08 (d, J = 4.3 Hz, 1H), 3.40 – 3.33 (m, 1H), 1.21 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 175.29, 134.65, 127.75, 124.44, 122.91, 120.71, 118.05, 117.22, 112.95, 74.29, 34.03, 15.35. NHOHO OHFNHOHO OHFNHOHO OHCl110 6Cl-11 (Appendix Figure B-17 and Appendix Figure B-18): 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 1.9 Hz, 1H), 7.18 (d, J = 2.3 Hz, 1H), 6.97 (dd, J = 8.4, 1.9 Hz, 1H), 4.12 (d, J = 4.5 Hz, 1H), 3.39 (qd, J = 7.0, 4.2 Hz, 1H), 1.23 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 175.76, 137.06, 126.05, 125.94, 124.19, 120.59, 119.04, 117.95, 111.50, 74.66, 34.54, 15.91. 7Cl-11 (Appendix Figure B-19 and Appendix Figure B-20): 1H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.21 (d, J = 2.5 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 6.98 (t, J = 7.8 Hz, 1H), 4.16 (d, J = 4.6 Hz, 1H), 3.41 (dt, J = 7.1, 3.8 Hz, 1H), 1.25 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 175.10, 132.82, 128.53, 123.82, 120.28, 119.26, 118.33, 117.73, 115.81, 73.97, 34.09, 15.44. 4.2.5 Synthesis of indolmycin and indolmycin derivatives The synthetic method for making 6 and its derivatives from 11 and its derivatives was adapted from literature methods to make 6 (Figure 4.2).58,60 To form the indolmycenic acid ethyl ester (13), or one of its derivatives, 85 µL of acetyl chloride was mixed into 300 µL of ethanol on ice for 30 min. Compound 11, or one of its derivatives, was dissolved in 200 µL of ethanol and added slowly to the acetyl chloride and ethanol mixture over 2-3 min. The resulting mixture was then stirred at room temperature for six hours. The solvent was rotary evaporated and the resulting residue was dissolved in 400 µL ethyl acetate, washed with 280 µL saturated sodium bicarbonate and 225 µL of brine, and then dried over anhydrous sodium sulfate before the solvent was speed vacuumed, leaving a residue of 13, or one of its derivatives. NHOHO OHClNHOHO OHCl111 The oxazolinone ring was cyclized to form N-desmethyl-indolmycin (14), or one of its derivatives, using 20 mg of guanidine, 20 mg of molecular sieves 3Å, and 240 µL of KOtBu (1 M in THF), which were mixed with 80 µL tert-Butanol at room temperature for three days. The residue of 13, or one of its derivatives, from the esterification step was dissolved in 160 µL of tert-butanol (also dried with sieves for three days for derivatives, but not for initial synthesis of 6) and added to the guanidine/sieves/KOtBu mixture, which was then mixed for 7.5 hours at room temperature. The reaction was stopped with 800 µL of cold, saturated ammonium chloride. The liquids partitioned into two layers, which were separated from each other after centrifugation. The aqueous ammonium chloride layer was washed with 500 µL of ethyl acetate, which was combined with the organic layer. Then the organic layer was washed with 200 µL of 5% sodium bicarbonate and dried with anhydrous sodium sulfate before the solvent was speed vacuumed, leaving a residue of 14, or one of its derivatives. Compound 6 and its derivatives were formed by an amine exchange of 14 and derivatives of 14 obtained from the previous reaction. Compound 14, or one of its derivatives, was dissolved in 65 µL of 40% (w/v) methylamine and incubated at 4°C for five hours. The solvent was evaporated by speed vacuum without heat and the resulting residue was dissolved in 500 µL of methanol for purification by semi-preparative HPLC, as described above for the purification of 11 and its derivatives. To further purify 6 from residual 11 after an initial semi-preparative HPLC purification, semi-preparative HPLC was run again, but at 48%:52% to 54%:46% HClEtOH(esterification)guanidine hydrochloride/tBuOK/tBuOH/mol. sieves 3Å40% MeNHH2O/211NHOHOHOX 13NHOHOOX14NHONONH2X 6NHONONHX5-28% yieldFigure 4.2: Synthetic scheme to 6 from 11, adapted from literature methods.58,60 112 ACN:water (v/v) with 0.1% (v/v) formic acid over 12 min. Fractions containing 6, or one of its derivatives, were verified by LC-MS and lyophilized to measure the final mass and determine the amount obtained. Compound 6 and its derivatives were characterized by HR-MS and NMR to confirm their identity. HR-MS was done on a Waters/Micromass LCT TOF-MS with electrospray ionization in positive ion mode. NMR analysis was done in DMSO-d6 with a Bruker Avance 600 MHz spectrometer or a Bruker Avance 400dir spectrometer. Tautomeric isomers of 6 and its derivatives were observed from all the NMR spectra in a 2:1 ratio, favouring the natural tautomer (Figure 4.3), which was confirmed by a varied temperature NMR experiment was done on 5F-6 between 25°C and 115°C with a Bruker Avance 400inv spectrometer (Appendix Figure B-31). Observation of these isomers has been reported previously, also in a 2:1 ratio favouring the natural tautomer, however the bioactivity of the non-natural tautomer is not known as the individual tautomers have not been isolated..31,58,60 6 (Appendix Figure B-21, Appendix Figure B-22, Appendix Figure B-23, Appendix Figure B-24 and Appendix Figure B-25): 1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 8.75 (s, 1/3H), 8.68 – 8.60 (m, 2/3H), 7.58 (dd, J = 8.0, 4.3 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 2.4 Hz, 1/3H), 7.15 (d, J = 2.4 Hz, 2/3H), 7.08 (ddt, J = 9.1, 7.8, 1.7 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 4.94 (d, J = 2.6 Hz, 2/3H), 4.91 (d, J = 2.7 Hz, 1/3H), 3.66 – 3.51 (m, 1H), 2.80 (d, J = 4.5 Hz, 2H), 2.77 (s, 1H), 1.25 (d, J = 7.3 Hz, 1H), 1.19 (d, J = 7.1 Hz, 2H). 13C NMR (101 NHOONNHNHOONNHNHOONHNFigure 4.3: Predicted tautomeric isomers of indolmycin observed from 1H NMR spectra. The tautomer on the left is the natural tautomer. 113 MHz, DMSO-d6) δ 186.59, 175.68, 136.20, 126.07, 122.44, 121.01, 118.52, 118.46, 115.32, 111.46, 85.35, 31.69, 28.81, 13.56. 5F-6 (Appendix Figure B-26, Appendix Figure B-27, Appendix Figure B-28, Appendix Figure B-29, Appendix Figure B-30 and Appendix Figure B-31): 1H NMR (400 MHz, DMSO-d6) δ 11.02 (d, J = 3.1 Hz, 1H), 8.70 (s, 1/3H), 8.67 – 8.59 (m, 2/3H), 7.38 – 7.19 (m, 3H), 6.96 – 6.85 (m, 1H), 4.93 (d, J = 2.7 Hz, 2/3H), 4.90 (d, J = 2.8 Hz, 1/3H), 3.63 – 3.47 (m, 1H), 2.79 (d, J = 4.4 Hz, 2H), 2.77 (s, 1H), 1.25 (d, J = 7.2 Hz, 1H), 1.19 (d, J = 7.2 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 187.28, 176.33, 157.27 (d, J = 230.8 Hz), 133.42 (d, J = 8.2 Hz), 126.92 (d, J = 9.8 Hz), 125.24, 116.18 (d, J = 4.9 Hz), 112.97 (d, J = 9.6 Hz), 109.75 (d, J = 26.1 Hz), 104.02 (d, J = 23.4 Hz), 85.84, 32.29, 29.42, 14.32. 6F-6 (Appendix Figure B-32 and Appendix Figure B-33): 1H NMR (600 MHz, DMSO-d6) δ 10.97 (s, 1H), 8.69 (s, 1/3H), 8.64 – 8.59 (m, 2/3H), 7.57 – 7.51 (m, 1H), 7.18 – 7.05 (m, 2H), 6.85 – 6.77 (m, 1H), 4.89 (d, J = 2.7 Hz, 2/3H), 4.86 (d, J = 2.8 Hz, 1/3H), 3.60 – 3.48 (m, 1H), 2.76 (d, J = 4.7 Hz, 2H), 2.73 (d, J = 4.1 Hz, 1H), 1.20 (d, J = 7.2 Hz, 1H), 1.14 (d, J = 7.0 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 186.67, 175.76, 158.79 (d, J = 234.0 Hz), 136.04 (d, J = 12.7 Hz), 123.09 (d, J = 3.3 Hz), 122.98, 119.68 (d, J = 10.1 Hz), 115.58, 106.91 (d, J = 24.3 Hz), 97.36 (d, J = 25.3 Hz), 85.25, 31.67, 28.83, 13.62. 7F-6 (Appendix Figure B-34 and Appendix Figure B-35): 1H NMR (600 MHz, DMSO-d6) δ 11.44 (s, 1H), 8.73 (s, 1/3H), 8.68 – 8.64 (m, 2/3H), 7.46 – 7.32 (m, 1H), 7.27 – 7.14 (m, 1H), 7.01 – 6.88 (m, 2H), 4.94 (d, J = 2.6 Hz, 2/3H), 4.90 (d, J = 2.8 Hz, 1/3H), 3.65 – 3.51 (m, 1H), 2.79 (d, J = 4.6 Hz, 2H), 2.76 (s, 1H), 1.25 (d, J = 7.2 Hz, 1H), 1.18 (d, J = 7.1 Hz, NHOONNHFNHOONNHFNHOONFNH114 2H). 13C NMR (151 MHz, DMSO-d6) δ 186.64, 175.77, 149.25 (d, J = 242.6 Hz), 130.15 (d, J = 5.7 Hz), 123.85 (d, J = 19.9 Hz), 123.69, 118.83 (d, J = 6.2 Hz), 116.56, 114.92, 105.88 (d, J = 16.0 Hz), 85.16, 31.71, 28.84, 13.67. 5Cl-6 (Appendix Figure B-36 and Appendix Figure B-37): 1H NMR (600 MHz, DMSO-d6) δ 11.14 (s, 1H), 8.70 (s, 1/3H), 8.67 – 8.62 (m, 2/3H), 7.64 – 7.57 (m, 1H), 7.39 – 7.31 (m, 1H), 7.27 – 7.20 (m, 1H), 7.10 – 7.01 (m, 1H), 4.94 (d, J = 2.8 Hz, 2/3H), 4.89 (d, J = 2.8 Hz, 1/3H), 3.64 – 3.51 (m, 1H), 2.81 – 2.76 (m, 3H), 1.27 (d, J = 7.2 Hz, 1H), 1.18 (d, J = 7.1 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 186.66, 175.72, 134.62, 127.24, 124.42, 123.23, 121.02, 117.82, 115.21, 113.01, 85.13, 31.51, 28.84, 13.76. 6Cl-6 (Appendix Figure B-38 and Appendix Figure B-39): 1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.70 (s, 1/3H), 8.65 – 8.59 (m, 2/3H), 7.64 – 7.51 (m, 1H), 7.42 – 7.30 (m, 1H), 7.25 – 7.10 (m, 1H), 7.04 – 6.91 (m, 1H), 4.92 (d, J = 2.7 Hz, 2/3H), 4.89 (d, J = 3.0 Hz, 1/3H), 3.63 – 3.48 (m, 1H), 2.78 (d, J = 4.1 Hz, 2H), 2.76 (s, 1H), 1.25 (d, J = 7.2 Hz, 1H), 1.19 (d, J = 7.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 186.58, 175.71, 136.56, 125.74, 124.93, 123.65, 120.06, 118.75, 115.60, 111.02, 85.17, 31.60, 28.80, 13.63. 7Cl-6 (Appendix Figure B-40 and Appendix Figure B-41): 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.69 – 8.63 (m, 1H), 7.62 – 7.50 (m, 1H), 7.26 – 7.09 (m, 2H), 7.05 – 6.95 (m, 1H), 4.94 (d, J = 2.7 Hz, 2/3H), 4.91 (d, J = 2.8 Hz, 1/3H), 3.67 – 3.48 (m, 1H), 2.79 (d, J = 4.2 Hz, 2H), 2.75 (s, 1H), 1.27 (d, J = 7.2 Hz, 1H), 1.19 (d, J = 7.2 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 186.59, 175.73, 132.92, 128.12, 123.81, 120.58, 119.55, 117.81, 116.78, 115.85, 85.14, 31.70, 28.82, 13.81. NHOONNHClNHOONNHClNHOONNHCl115 4.2.6 Bioactivity assays9 Disk diffusion assays were done on Mueller-Hinton Agar plates with MRSA (Staphylococcus aureus ATCC 33591). Each derivative was dissolved in water with 0.5% DMSO and deposited on the disks. Rifampicin (1 µg) was used a positive control and 0.5% DMSO was used as a negative control. Each disk contained either 10 µg of 6, 20 µg of 6F-6 or 7F-6, or 30 µg of 5F-6, 5Cl-6, 6Cl-6, or 7Cl-6 and was deposited onto the petri dishes containing a lawn of the MRSA culture. The petri dishes were allowed to grow overnight, after which zones of inhibition were visible. The minimum inhibitory concentration (MIC) measurements were done by diluting an overnight culture of S. aureus ATCC 33591 to ~ 1 x 105 cells/mL with Mueller-Hinton Broth (MHB). Various dilutions of 1 and its derivatives and the positive control, rifampicin, were prepared. DMSO was used as a negative control. Concentrations ranged from 50 µg/mL to 0.128 ng/mL for 1, 5F-6, 6F-6 and 7F-6 and from 200 µg/mL to 0.512 ng/mL for 5Cl-6, 6Cl-6 and 7Cl-6. The rifampicin sample concentrations ranged from 10 µg/mL to 0.0256 ng/mL. The 100 µL of S. aureus cells and 10 µL of each diluted sample were combined in a 96-well plate and diluted to 200 µL with 90 µL MHB. Each sample concentration for each compound was tested in triplicate. The 96-well plate was covered and incubated at 37°C for 18 h before measuring the OD at 600 nm using a Molecular Devices FilterMax F5 Multi-Mode Microplate Reader. Readings from blank samples were subtracted from each sample reading to remove background signal and the percent of cells alive was calculated in comparison to the DMSO negative control. The MIC50 values were determined from the percent alive using the Quest Graph IC50 Calculator online tool from AAT Bioquest.226 Docking experiments to test analog binding to a TrpRS was done using AutoDock Vina194 and the structures for 6 and its derivatives were generated using Phenix eLBOW.178 The TrpRS model used for docking was from Bacillus stearothermophilus (PDB: 5DK4) and the cocrystallized ligands, ATP, Mg2+ and indolmycin, were removed from the model before docking. 9 Disk diffusion and MIC assays were performed by J. C. 116 4.3 Results 4.3.1 Production of indolmycin 4.3.1.1 Heterologous expression of indolmycin genes Armed with the previously elucidated biosynthetic pathway for 6, an attempt to create an in vivo system to make 6 in E. coli was made. First, all necessary genes were cloned into four plasmids and were co-expressed in E. coli BL21 (DE3) (Figure 4.4). This strain was given the name E. coli I1234670P5 (Table 4.1). The genes needed to produce indolmycin in E. coli were ind1, ind2, ind3, ind4, ind6, ind7, ind0 and pel5, a gene homologous to ind5 from Paenibacillus elgii B69.113,174 Unfortunately, only a small amount of 6 ([M+H]+ = 258 m/z; Figure 4.5) was produced (~170 µg/L of culture) and could not be improved (Figure 4.6). However, this construct did produce more substantial amounts of 11 ([M+H]+ = 220 m/z; Figure 4.5) at 40-50 mg/L of culture, along with a shunt product, C-desmethyl-indolmycenic acid (12; [M+H]+ = 206 m/z; Figure 4.5). cpel5RT7pP5I0COLAind0RT7 ind1RT7pI1I2ETind2RT7 ind3RT7pI3I4CDFind4RT7 ind6RT7pI6I7ACYCind7RT7atrpSRT7pTSI0COLAind0RT7b1 kbind0 ind1 ind2 ind3 ind4 ind6 ind71 kb1.5 kb0.75 kbpel51 kbtrpS2 kb2.5 kbFigure 4.4: Cloning genes for in vivo production of 6, 11 and derivatives of 11. (a) Plasmids made and used in E. coli I1234670P5 to make 6 in vivo. (b) Plasmid used in E. coli I1234670TS to replace pP5I0COLA from E. coli I1234670P5 for production of 6 and its derivatives. (c) Agarose gels of PCR amplifications for each gene cloned into each of the five plasmids. Expected sizes for each gene are ind0: 1018 base pairs; ind1: 1032 base pairs; ind2: 1761 base pairs; ind3: 1406 base pairs; ind4: 1198 base pairs; ind6: 737 base pairs; ind7: 760 base pairs; pel5: 1011 base pairs; trpS: 2050 base pairs. 117 11: X = H [M+H]+ 220 m/zF-11: X = F [M+H]+ 238 m/zCl-11: X = Cl [M+H]+ 254/256 m/zBr-11: X = Br [M+H]+ 298/300 m/zOH-11: X = OH [M+H]+ 236 m/zaza-11: X = H; azaindole [M+H]+ 221 m/zNHOHOHOX13: X = H [M+H]+ 248 m/zF-13: X = F [M+H]+ 266 m/zCl-13: X = Cl [M+H]+ 282/284 m/zNHOHOOX14: X = H [M+H]+ 244 m/zF-14: X = F [M+H]+ 262 m/zCl-14: X = Cl [M+H]+ 278/280 m/zNHONONH2X6: X = H [M+H]+ 258 m/zF-6: X = F [M+H]+ 276 m/zCl-6: X = Cl [M+H]+ 292/294 m/zNHONONHX12: X = H [M+H]+ 206 m/zF-12: X = F [M+H]+ 224 m/zCl-12: X = Cl [M+H]+ 240/242 m/zBr-12: X = Br [M+H]+ 284/286 m/zOH-12: X = OH [M+H]+ 222 m/zaza-12: X = H; azaindole [M+H]+ 207 m/zNHOHO OHXFigure 4.5: Summary of compound [M+H]+ ions. 118 4.3.1.2 Indolmycenic acid purification and indolmycin synthesis Compound 11 has often been a focus of total synthetic efforts towards 6, as it is a key chiral intermediate.31,55–60 From 11, a common method of producing indolmycin involved a three-step synthesis, in which 11 was esterified, the oxazolinone ring was cyclized using guanidine, and the exocyclic nitrogen was methylated (Figure 4.2).58,60 Since production of 11 was much higher than for 6 from E. coli I1234670P5, a semi-synthetic method of obtaining 6 was pursued for use in a three-step synthesis to produce 6. Now focused on 11 rather than 6, an attempt was made to remove extraneous genes from the biosynthetic platform but was unsuccessful and the changes resulted in reduced amounts of 11 (Figure 4.7). Therefore, the full four-gene construct was employed toward production of 11. Then, the three-step synthesis was used (Figure 4.2),58,60 in which purified 11 was esterified to make the ethyl ester (13; [M+H]+ = 248 m/z; Figure 4.5), cyclized to give N-desmethyl-indolmycin (14; [M+H]+ = 244 m/z; Figure 4.5), and methylated at the exocyclic nitrogen to give 6 ([M+H]+ = 258 m/z; Figure 4.5) (Figure 4.8). A total of 2.6 mg of 6 was made with 5.0% yield from 11. The low yield may be attributed to a high amount of 11 regenerated in the oxazolinone-cyclization step (Figure 4.8b and Figure 4.9), likely due to insufficient drying of the tert-butanol solvent. 6 12 11 4 5 6 7 8 9 10 11 12Retention Time (min)indolmycin standard 206 m/z 220 m/z 258 m/z Figure 4.6: LC-MS analysis of observed products from E. coli I1234670P5. Peaks are labelled with the corresponding compound number and shown in a coloured box. 119 15 20Retention Time (min)E. coli I120E. coli I1234670P5206 m/z 220 m/z 12 11Figure 4.7: HPLC comparison of E. coli I120 and E. coli I1234670P5 metabolites to understand the effect of removing genes presumably not needed for production of 11. Relevant peaks identified as indolmycin pathway metabolites are indicated with a coloured box, compound number and their corresponding m/z from LC-MS analysis. 120 10 12.5 157.5 17.5 20Retention Time (min)0 hours6 hours220 m/z 248 m/z 11 13a 244 m/z 248 m/z 220 m/z 10 12.5 157.5 17.5 20Retention Time (min)131114b 220 m/z 258 m/z Retention Time (min)10 12.57.511 6c 220 m/z 258 m/z 10 12.57.5Retention Time (min)11 6d Figure 4.8: LC-MS analysis for synthesis and purification of 6. (a) Total ion chromatograms of the esterification at the beginning (0 hours) and the end (6 hours) of the reaction. (b) Total ion chromatograms of the oxazolinone-cyclization reaction. (c) Total ion chromatogram of the N-methylation reaction after initial purification by semi-preparative HPLC. (d) Total ion chromatogram of 6 after final purification by semi-preparative HPLC. Compounds are indicated by a coloured box and numbered. [M+H]+ signals corresponding to each observed compound are indicated beside each coloured box. 121 Table 4.3: Amounts of 11-derivatives and 6-derivatives obtained. Yields given are for synthetic steps from 11 only. Amounts of 11 are from 1 L of bacterial culture. Low yield for underivatized 6 is attributed to improper drying of solvent during production of 14. Synthetic scheme is given in Figure 4.2. 4.3.2 Production of indolmycin derivatives 4.3.2.1 Use of TrpS for improved indole incorporation In order to make derivatives of 6, it was desirable to feed indole-substituted precursors, such as derivatives of 8, to E. coli I1234670P5. However, the lack of accessible derivatives of 8 inspired the use of indole derivatives, which are more widely accessible. The TrpS from S. enterica has been previously shown to be able to accept a wide variety of indole derivatives and convert them to derivatives of 8, in vivo, using L-serine.88 This TrpS was incorporated into the biosynthetic platform to make derivatives of 11 to be used as precursors to synthesize derivatives 11 and derivatives (mg) 6 and derivatives (mg) % yield indole 44.1 2.6 5.0 5-fluoroindole 26.9 7.8 25 6-fluoroindole 15.1 2.6 15 7-fluoroindole 24.9 3.1 11 5-chloroindole 9.1 1.7 16 6-chloroindole 10.3 3.3 28 7-chloroindole 10.5 2.6 22 10 12.57.5Retention Time (min)11 6b 10 12.5 157.5 17.5Retention Time (min)131114a Figure 4.9: UV-Vis traces obtained during LC-MS analysis of synthetic steps showing regeneration of 11. Absorbance measurements were made at 280 nm. Traces show high levels of 11 regenerated after the oxazolinone-cyclization step in (a) and high levels of 11 remaining after N-methylation reaction in (b). 122 of 6. pel5 was substituted for trpS in this biosynthetic platform (Figure 4.4b), which was subsequently named E. coli I1234670TS. Improved production of fluorinated metabolites, 5-fluoro-indolmycenic acid (5F-11; [M+H]+ = 238 m/z; Figure 4.5) and 5-fluoro-C-desmethyl-indomycenic acid (5F-12; [M+H]+ = 224 m/z; Figure 4.5), was observed from E. coli I1234670TS fed 5-fluoroindole (Figure 4.10a). 5-fluoroindole was also used to optimize the feeding conditions, which showed that 5F-11 amounts were optimal when fed 0.5 mM of 5-fluoroindole per day over two days (Figure 4.11). NHFNHClNHOHNHFNHF NHFNHClNHCl NHClNHHONHHO NHBrNNHNNHN NH NNHb 10 15 20no trpSRetention Time (min)trpStrpStrpSno trpSno trpSno trpStrpSa206 m/z224 m/z220 m/z238 m/z125F-12 115F-11Figure 4.10: TrpS addition to the biosynthetic platform allows incorporation of substituted indoles into 11. (a) LC-MS analysis of strains with and without trpS (E. coli I1234670TS and E. coli I1234670P5, respectively) when fed 5-fluoroindole. Incorporation of fluorinated compounds into the 6-pathway metabolites (5F-12 is [M+H]+ = 224 m/z and 5F-11 is [M+H]+ = 238 m/z) is improved in E. coli I1234670TS. (b) Indole derivatives tested for incorporation by E. coli I1234670TS. Dark blue shows indoles that were detected and further purified, medium blue was possibly detected by LC-MS, but was not further verified, and light blue did not show detectable derivatives of 11. 123 4.3.2.2 Scope of indole incorporation To determine the scope of indole derivatives accepted by the biosynthetic platform, a variety of indoles were fed to E. coli I1234670TS and the production of 11 and its derivatives was observed. Out of the indoles tested, the fluorinated and chlorinated indoles substituted at the 5-, 6- and 7-positions were the best accepted by the biosynthetic platform (Figure 4.10b and Figure 4.12 and Figure 4.13). Lower acceptance of indoles substituted at the 4-position may be due to a steric hindrance, as 4-fluoroindole was moderately accepted, while 4-chloroindole was not utilized at all. Although there was evidence that some of the azaindoles and hydroxyindoles were incorporated, further work would need to be done to confirm, optimize and scale up the purification of these compounds (Figure 4.13). It is possible that the hydroxyindoles and azaindoles were less accepted because the mechanism of indole transport into the cells may be disrupted by their different chemical properties or they may have bound unproductively in the active site of TrpS and were unable to be converted to derivatives of 8. Additionally, reduced nucleophilicity of the azaindoles in particular might have had an effect on the ability for TrpS to convert the indoles to derivatives of 8, as has been observed previously.85 020004000600080001000012000140001600018000200002.0 mM 1.0 mM 0.5 mM 0.25 mM 0.1 mM 0.05 mMPeak Area (mAU2 )Amount of 5F-indole fed per dayOne DayTwo DaysThree DaysFigure 4.11: HPLC analysis of 5F-11 amounts under different feeding conditions. Amount of 5F-11 was approximated based on peak area measured at 280 nm. Amount of 5F-11 produced for 2.0 mM and 1.0 mM was only tested on the first day. 124 10 15 204-fluoroRetention Time (min)238 m/z224 m/z220 m/z206 m/z10 15 206-fluoroRetention Time (min)238 m/z224 m/z220 m/z206 m/z10 15 207-fluoroRetention Time (min)238 m/z224 m/z220 m/z206 m/z10 15 20 25 30242 m/z240 m/z220 m/z206 m/z254 m/z256 m/z4-chloroRetention Time (min)10 15 20 25 30242 m/z240 m/z220 m/z206 m/z254 m/z256 m/z5-chloroRetention Time (min)10 15 20 25 30242 m/z240 m/z220 m/z206 m/z254 m/z256 m/z6-chloroRetention Time (min)10 15 20 25 30242 m/z240 m/z220 m/z206 m/z254 m/z256 m/z7-chloroRetention Time (min)10 15 20 25 30Retention Time (min)5-bromo286 m/z284 m/z220 m/z206 m/z298 m/z300 m/zFigure 4.12: LC-MS analysis of halogen-substituted indole incorporation into 11 using E. coli I1234670TS. Each trace represents an extracted ion chromatogram of [M+H]+ signals for 12, 11, substituted 12 and substituted 11. 125 10 15 205Retention Time (min)4-azaindole 221 m/z207 m/z220 m/z206 m/z10 15 205Retention Time (min)5-azaindole 221 m/z207 m/z220 m/z206 m/z10 15 205Retention Time (min)7-azaindole221 m/z207 m/z220 m/z206 m/z10 15 205Retention Time (min)4-hydroxyindole236 m/z222 m/z220 m/z206 m/z10 15 205Retention Time (min)5-hydroxyindole236 m/z222 m/z220 m/z206 m/z10 15 205Retention Time (min)6-hydroxyindole236 m/z222 m/z220 m/z206 m/z6-azaindoleRetention Time (min)10 15 205221 m/z207 m/z220 m/z206 m/zFigure 4.13: LC-MS analysis of hydroxyindole or azaindole incorporation into 11 using E. coli I1234670TS. Each trace represents an extracted ion chromatogram of [M+H]+ signals for 12, 11, derivatives of 12 and derivatives of 11. 7-azaindole, 5-hydroxyindole and 6-hydroxyindole show some evidence of incorporation. 126 4.3.2.3 Production of indolmycin derivatives Feeding of fluoro and chloroindoles substituted at the 5-, 6- and 7-positions was further scaled up for purification of 11-derivatives and downstream synthesis of 6-derivatives (Figure 4.14, Figure 4.15, Figure 4.16 and Figure 4.17). Each purified derivative of 11 and 6 was characterized by HR-MS and NMR (Table 4.4 and Appendix C). Cultures fed 5-fluoroindole produced the highest amount of any derivative of 11 with 26.9 mg of 5F-11, but still lower than cultures fed indole, which produced 44.1 mg of 11 (Table 4.3). Overall, cultures fed the fluorinated indoles produced a higher amount of 11-derivatives than the cultures fed with the chlorinated indoles. This result indicates that the larger substitution of the chlorine atom, compared to the fluorine atom, may be causing steric hindrance in some of the enzyme active sites, which reduces the efficiency of turnover. Additionally, growth and feeding conditions were optimized for 5-fluoroindole, so they may have biased the results towards obtaining more 5F-11 than other derivatives of 11. Derivatives of 6 were made with between 11% and 28% yield from the three-step synthesis (Figure 4.2) and between 1.7 mg and 7.8 mg of each derivative was obtained (Table 4.3). These improvements over the yield obtained for underivatized-6 can be attributed to improvements in drying tert-butanol in the oxazolinone-cyclization step, leading to less regeneration of 11 and its derivatives as side-products. 127 Derivativesof 11NHOHO OHNHOHO OHFNHOHO OHClNHOHO OHFNHOHO OHFNHOHO OHClNHOHO OHClDerivativesof 6NHOONNHNHOONNHFNHOONNHClNHOONNHFNHOONNHFNHOONNHClNHOONNHClTrpSInd2Ind1NHNH3O OHNHNH2O OHFNHNH2O OHClNHNH2O OHF NHNH2O OHClNHNH2O OHFNHNH2O OHClDerivativesof 8Indolederivatives NHNHFNHClNHF NHClNHFNHClPurificationand SynthesisNHNH2O OHNHOHO OH E. coliI1234670TSFigure 4.14: Semi-synthetic scheme of producing 6 and its derivatives starting from indole derivatives. 8, 11 and their derivatives are produced biosynthetically, in vivo, from E. coli I1234670TS. Compound 11 and derivatives of 11 are purified and synthesized to make 6 and its derivatives. 128 238 m/z 266 m/z 10 12.5 157.5 17.5 20Retention Time (min)0 hours6 hours5-fluoro Retention Time (min)10 12.5 157.5 17.5 206-fluoro 238 m/z 266 m/z 0 hours6 hours238 m/z 266 m/z 10 12.5 157.5 17.5 20Retention Time (min)0 hours6 hours7-fluoro 254/256 m/z 282/284 m/z 10 12.5 157.5 17.5 20Retention Time (min)0 hours6 hours6-chloro 10 12.5 157.5 17.5 20Retention Time (min)0 hours6 hours5-chloro 254/256 m/z 282/284 m/z 254/256 m/z 282/284 m/z 10 12.5 157.5 17.5 20Retention Time (min)0 hours6 hours7-chloro Figure 4.15: LC-MS analysis of 11-derivative esterification. Total ion chromatograms are shown for each derivative (5-fluoro, 6-fluoro, 7-fluoro, 5-chloro, 6-chloro and 7-chloro) at the beginning of the reaction, immediately after mixing reagents (0 hours) and the end of the reaction (6 hours). The starting materials, 11-derivatives, and the products, 13-derivatives, are indicated by a blue or green box, respectively. The m/z for each compound is given above the corresponding boxes. 129 262 m/z 238 m/z 10 12.5 157.5 17.5 20Retention Time (min)5-fluoro 262 m/z 238 m/z 10 12.5 157.5 17.5 20Retention Time (min)6-fluoro 262 m/z 238 m/z 10 12.5 157.5 17.5 20Retention Time (min)7-fluoro 278/280 m/z 254/254 m/z 10 12.5 157.5 17.5 20Retention Time (min)5-chloro 278/280 m/z 254/254 m/z 10 12.5 157.5 17.5 20Retention Time (min)6-chloro 278/280 m/z 254/254 m/z 10 12.5 157.5 17.5 20Retention Time (min)7-chloro Figure 4.16: LC-MS analysis of 13-derivative oxazolinone-cyclizations. Total ion chromatograms are shown for each derivative (5-fluoro, 6-fluoro, 7-fluoro, 5-chloro, 6-chloro and 7-chloro). Oxazolinone-cyclized products, 14-derivatives, are indicated with a purple box and 11-derivative side-products generated during the reaction are shown with a blue box. The m/z for each compound is indicated. 130 276 m/z 10 12.57.5Retention Time (min)5-fluoro 276 m/z 10 12.57.5Retention Time (min)6-fluoro 276 m/z 10 12.57.5Retention Time (min)7-fluoro 292/294 m/z 10 12.57.5Retention Time (min)5-chloro 292/294 m/z 10 12.57.5Retention Time (min)6-chloro 292/294 m/z 10 12.57.5Retention Time (min)7-chloro Figure 4.17: LC-MS analysis of 14-derivative N-methylation. Total ion chromatograms are shown for each derivative (5-fluoro, 6-fluoro, 7-fluoro, 5-chloro, 6-chloro and 7-chloro) after semi-preparative HPLC purification. 6-derivative products are indicated with a pink box. The m/z for each product is indicated. 131 Table 4.4: HR-MS analysis of purified 11, 6 and their derivatives. Observed Mass (m/z) Calculated Mass (m/z) Chemical Formula Error (ppm) 11 219.0906 219.0895 C12H13NO3 -5 6 257.1167 257.1164 C14H15N3O2 -1 5F-11 237.0808 237.0801 C12H12FNO3 -3 5F-6 275.1082 275.1070 C14H14FN3O2 -5 6F-11 237.0809 237.0801 C12H12FNO3 -3 6F-6 275.1078 275.1070 C14H14FN3O2 -3 7F-11 237.0811 237.0801 C12H12FNO3 -4 7F-6 275.1076 275.1070 C14H14FN3O2 -2 5Cl-11 253.0514 253.0506 C12H12ClNO3 -3 5Cl-6 291.0777 291.0775 C14H14ClN3O2 -1 6Cl-11 253.0504 253.0506 C12H12ClNO3 -1 6Cl-6 291.0784 291.0075 C14H14ClN3O2 -3 7Cl-11 253.0504 253.0506 C12H12ClNO3 -1 7Cl-6 291.0785 291.0075 C14H14ClN3O2 -3 4.3.3 Bioactivity against MRSA Finally, the bioactivities of 6 and its derivatives were tested with a disk diffusion assay against MRSA (Figure 4.18). Compound 6 showed the highest activity with a zone of inhibition visible when 10 µg of the compound was loaded onto each disk. 6F-6 and 7F-6 showed a zone of inhibition at 20 µg per disk while 5F-6 showed inhibition at 30 µg per disk. The chlorinated derivatives of 6 did not show any bioactivity at the maximum amount tested in the disk diffusion assay (30 µg). MIC50 values were determined for each compound (Table 4.5). The MIC50 values demonstrate 6 is a more potent inhibitor of MRSA than its derivatives, while 6F-6 showed the most potent inhibition of MRSA of any of the derivatives, followed by 7F-6 and 5F-6. The lack of bioactivity shown by the chlorinated compounds may be due to the bulky chlorinated substituent hindering the compounds’ abilities to bind to the TrpRS target, which is supported by docking the analogs into a bacterial TrpRS structure (Figure 4.19). However, bioactivity of the fluorinated substituents implies that 4F-6 could also show activity if its production was to be optimized in the future. 132 rifampicinDMSOindolmycinrifampicinrifampicin rifampicinDMSODMSODMSO6F-indolmycin7F-indolmycin5F-indolmycin5Cl-indolmycin6Cl-indolmycin7Cl-indolmycinFigure 4.18: Disk diffusion assay of 6 and its derivatives against MRSA. Rifampicin (1 µg) and DMSO (0.5%) were used as positive and negative controls, respectively. 10 µg of 6, 20 µg of 6F-6 and 7F-6 and 30 µg of 5F-6, 5Cl-6, 6Cl-6 and 7Cl-6 were loaded onto each disk. 133 Table 4.5: MIC50 values determined for 6 and its derivatives against MRSA. Values represent the average of three replicates ± the standard deviation. For 6, 5F-6, 6F-6 and 7F-6, concentration ranges between 50 µg/mL to 0.128 ng/mL were tested, and for 5Cl-6, 6Cl-6 and 7Cl-6, concentration ranges between from 200 µg/mL to 0.512 ng/mL were tested. Rifampicin was used as a positive control. MIC50 (µg/mL) rifampicin 0.0047 ± 0.0013 6 1.21 ± 0.04 5F-6 32.5 ± 19.6 6F-6 6.49 ± 0.03 7F-6 16.7 ± 4.2 5Cl-6 > 200 6Cl-6 > 200 7Cl-6 > 200 134 4.4 Discussion By combining biosynthetic and synthetic methods of making 6, a variety of novel halogenated derivatives of 6 were able to be made, some of which showed bioactivity against MRSA. Attempts of producing derivatives of 6 by either synthesis or biosynthesis have been indolmycin 5F-indolmycin6F-indolmycin 7F-indolmycin5Cl-indolmycin 6Cl-indolmycin7Cl-indolmycinFigure 4.19: Docking of indolmycin derivatives made in this study into an indolmycin-bound crystal structure of TrpRS from Bacillus stearothermophilus (PDB: 5DK4). Docked molecules (magenta) are compared with active site-bound indolmycin (white) from the crystal structure. Binding modes for the indole rings of chlorinated analogs could not be matched with the crystal structure. Oxazolinone conformations for fluorinated compounds may have differed from the crystal structure because other ligands in the active site which interact with the oxazolinone ring, including ATP and Mg2+, were not included in the docking model. 135 made previously. 5-hydroxy and 5-methoxy derivatives of 6 were observed when S. griseus ATCC12648 was fed the corresponding derivatives of indole or 8, but derivatives of 8 and indole substituted at the 6-position were not converted to 6-substituted derivatives of 6.67 However, incorporation of indoles substituted at the 6-position was observed in the E. coli I1234670TS system, indicating that the semi-synthetic method is able to overcome a limitation of the pure biosynthetic method associated with making 6-substituted derivatives. The enzymes causing this limitation in the natural pathway are likely to be found in Part III of 6 biosynthesis (Figure 4.1) because the limitation must come later than Ind1 and Ind2, which are found in Part II. Additionally, purely biosynthetic methods of obtaining 6 and these 5-hydroxy- and 5-methyoxy-derivatives has relied on Streptomyces sp. to produce the compounds, which has more limited potential for genetic tailoring than E. coli and is also more difficult to grow and maintain. Therefore, the E. coli I1234670TS system provides a more versatile method of production by using a more adaptable organism and overcoming natural substrate selectivity by only using two of the indolmycin biosynthetic enzymes. While the synthetic part of the semi-synthetic method helped to overcome biosynthetic challenges, the biosynthetic portion of the system also overcomes challenges associated with synthetic methods. Synthetic studies were often associated with making the chiral precursor, 11, through methods such as chiral auxiliary usage,59 epoxide ring opening,56,58 lipase-assisted enantioselective acetylation,60 or a combination of these methods.57 The E. coli I1234670TS biosynthetic method of obtaining 6 and its derivatives offers a simple alternative to synthetic methods by installing chirality in vivo. Derivatives made during synthetic efforts were focused on making known metabolites58 or the 5-methoxy-derivative,60 previously shown by Werner and Demain to have increased bioactivity.67 Attempts to synthesize other derivatives, particularly halogenated compounds, were likely not feasible as the methods employed to make these derivatized compounds, such as Larock indole synthesis,60,61 would be incompatible with halogen substitutions. Therefore, the E. coli I1234670TS biosynthetic method of making 11 overcomes the incompatibility of halogenated compounds with synthetic methods for making derivatives of 11, allowing a variety of novel halogenated derivatives of 6 to be made. The obtained derivatives were consistent with the initial reports of the promiscuous TrpS from S. enterica, which showed the lowest acceptance for 4-chloro, 5-chloro and 5-bromoindoles.88 While 4-chloro and 5-bromoindoles were not carried forward to larger-scale 136 growth and purification, the 5-chloroindole feeding resulted in the lowest amount of purified 11. Since low amounts of 4-fluoroindole incorporation were observed despite Goss’s observation of moderate incorporation, it is likely the indolmycin genes themselves that are sensitive to acceptance of substituents at this position. Azatryptophans have been previously made by TrpS but required longer reaction times due to reduced nucleophilicity,85,227 which explains why they may not have been incorporated by the semi-synthetic system if they were not given a long enough reaction time. Winn et al. (2018) was able to produce azaindole derivatives of the agrochemical, thaxtomin, using the S. enterica TrpS;94 however, longer reaction times were used (72 hours to make Trp derivatives and 5-7 days to make thaxtomin derivatives). While hydroxyindoles were not converted to derivatives of 11, hydroxytryptophans have been produced by the S. enterica TrpS in vitro;85 however, there are no examples of them being made in vivo. Several other indoles that have been successfully utilized by the S. enterica TrpS remain to be tested for acceptance by the semi-synthetic platform, including nitro-, methyl-, ethenyl-, ethynyl-, amino-, methoxy- and dual-substituted indoles,87–89,94 which also have the potential to show interesting bioactivities. Overall, the substrate acceptance and amounts of 11-derivatives obtained could be further improved with optimized reaction and purification conditions. Additionally, strains could be engineered to reduce endogenous tryptophan and enzymes, such as TrpS and Ind1/Ind2, could be engineering to improve the substrate scope. TrpS has already been the target of many enzyme engineering studies focused on improving the substrate scope;83,90,228–232 however, other TrpS variants and homologs were not used for this biosynthetic platform because they were demonstrated for in vitro use with purified enzymes, rather than in vivo. Compound 6 and the fluorinated derivatives of 6 showed bioactivity against MRSA with MIC values ranging from 1-33 µg/mL, demonstrating that these newly derivatized compounds may serve as useful molecules for development of new antibiotics. Observed bioactivity from the fluorinated compounds indicates that it may be worthwhile to produce and test other derivatives of 6. Additionally, the fluorinated compounds could be tested against more of 6’s identified targets, such as H. pylori,32 and P. falciparum,39 to see if the compounds show maintained, improved or reduced bioactivity. These compounds may also have other useful qualities that remain to be tested, including overcoming the narrow spectrum of activity or the hepatic toxicity of the original compound, which caused original development of 6 to be discontinued during the golden era of antibiotic discovery. 137 Here, the indolmycin biosynthetic enzymes were used in a semi-synthetic process to make a variety of 6-derivatives from substituted indoles. This platform applies the best parts of the biosynthetic and synthetic methods towards 6, while simultaneously pairing them with TrpS to open the door to several new indole-substituted analogs. The biosynthetic aspects of the process allowed for production of 11, and its derivatives, whose construction was often the aim of synthetic studies focused on making 6 and its derivatives. The synthetic aspects of the process allowed for a greater versatility of derivatives to be explored that the purely biosynthetic system may not have been able to produce, since previous studies indicate that some enzymes from the indolmycin biosynthetic pathway may be selective against indole-substitution at some positions. Overall, this work lays a foundation for making derivatives of 6, and perhaps analogs of other 8-derived molecules, to be tested for antimicrobial properties. This work also demonstrates the value of coupling synthetic and biosynthetic methods to make and diversify natural product-derived compounds, beyond just those useful as antimicrobials. 138 Chapter 5: Conclusions and future directions 5.1 General Conclusions The exploration of natural product biosynthesis is an important endeavor towards building industrially useful biocatalysts. By understanding how natural products are assembled, new enzymes and new ways of constructing molecules can be discovered that expand the known biocatalytic repertoire. In this work, the biosynthetic enzymes of indolmycin were shown to be useful biochemical tools to better understand the unique oxygen reactivity among some PLP-dependent enzymes and to produce halogenated indolmycin derivatives that were not easily accessible by chemical methods alone. Use of these enzymes as biochemical tools helps to expand the known the biocatalytic repertoire, which may contribute to more widespread use of biocatalysts to solve industrially relevant problems, such as more feasible production of natural products and their derivatives. During elucidation of indolmycin biosynthesis, a unique enzyme, known as Ind4, was discovered that catalyzes an oxygen- and PLP-dependent desaturation of L-arginine.113 An interest in studying the mechanism of Ind4, led to the discovery of a homologous, yet evolutionarily distinct enzyme, Plu4, which also catalyzes an oxygen- and PLP-dependent desaturation of L-arginine. The first crystal structure of an oxygen- and PLP-dependent desaturase, described in Chapter 2, revealed an active site that is highly similar to other previously studied oxygen- and PLP-dependent enzymes catalyzing L-arginine hydroxylation114–116 but with some evidence of different dynamics between the two types of enzymes. The crystal structures solved for Plu4 during different stages of catalysis aided mutagenesis studies to understand determinants for catalysis, including a His-Asp catalytic dyad. Although these crystal structures alone could not determine the cause for reactivity differentiation, particularly if the differences between the hydroxylases and desaturases are related to dynamics, this structural information is essential to gain the mechanistic knowledge needed for engineering biocatalysts.27 Therefore, the structural characterization described in Chapter 2 lays a foundation for potential engineering efforts that may be needed to make the arginine desaturases a useful biocatalytic tool. The biochemical and phylogenetic analysis of arginine desaturases in Chapter 3 supported the findings of the structural analysis in Chapter 2 by revealing that the hydroxylases and desaturases are more highly similar than previously expected. A minor hydroxylated side-139 product was observed from the desaturases suggesting that their mechanisms are closely related. An SSN analysis revealed that some desaturases are more closely related to a group of hydroxylases than they are to other desaturases and vice versa. EPR experiments and cytochrome c assays also suggested that superoxide is an intermediate in in the reaction for the first time in the oxygen- and PLP-dependent enzymes. This information, coupled with computational work, suggests that preorganization of water in the active site of the hydroxylases may be the cause for distinction between the two reactivities. Overall, this new structural and biochemical data has provided valuable information towards identifying a unified mechanism for the arginine oxidases and will aid efforts to engineer these enzymes. Additionally, the discovery and characterization of seven new desaturases will also aid in engineering efforts by providing a pool of candidates to use as starting points. In order to demonstrate a potential biocatalytic use for the indolmycin biosynthetic genes, in Chapter 4 the production of indolmycin and various indole-substituted derivatives was attempted. In doing so, the first halogenated derivatives of indolmycin were made using a semi-synthetic approach that overcame compatibility issues associated with solely synthetic methods previously used to make indolmycin. The fluorinated indolmycin compounds made using the semi-synthetic method showed promising bioactivity against MRSA, suggesting that they could be useful for clinical development. Although neither indolmycin nor its derivatives were produced using purely biosynthetic methods in vivo, the semi-synthetic approach demonstrated the utility that biocatalysts can have by supplementing current chemical processes to overcome challenges associated with a purely chemical method. Therefore, this work shows that the study of natural products and their biosynthesis can contribute to the expansion of the biocatalytic repertoire, allowing new molecules to be made, tested and used for industrial purposes. 5.2 Future Directions 5.2.1 Arginine oxidases Further studies regarding the arginine oxidases should be focused on understanding phylogenetic relationships, catalytic mechanisms and potential uses for the enzymes. Through SSN and genomic neighbourhood analysis and the discovery that the arginine oxidases can be organized into subgroups, it is clear that there are still many uncharacterized enzymes with potential arginine oxidase activity whose reactivity cannot be inferred from their genomic 140 neighbourhood. Many of these uncharacterized enzymes are a part of uncharacterized gene clusters, including the enzymes contained in non-indolmycin gene clusters in Group III and non-enduracididine gene clusters in Group II. Characterizing these unknown enzymes would serve several purposes. First, characterization would allow for further phylogenetic information and sub-group characterization. This information would contribute to a better understanding of how the hydroxylation, desaturation and perhaps overall oxygen reactivities naturally evolved by providing more sequence-activity relationship information. This information could improve the current understanding of how to engineer these reactivities into other enzymes. Second, this characterization would provide a larger pool of enzymes to draw from for future engineering efforts. Work to characterize more of these enzymes, including the work described in Chapter 3, would also provide clues to the function of the unknown gene clusters that encode some of these enzymes, which could lead to the discovery of new natural products and their biosynthesis. Although this work has contributed to a better understanding of the mechanism, there are still many mechanistic details regarding the arginine oxidases that should be elucidated before attempting engineering efforts toward industrial use. Based on these observations, a new hypothesis has been provided for differentiation of the hydroxylase and desaturase reactivities, which suggested that the dynamics of the enzyme or residues found outside the active site must be responsible for distinguishing the two reactivities, perhaps through manipulating preoganization of water. However, this hypothesis should be studied in more detail to determine its validity. This hypothesis could be investigated through molecular dynamics (MD) simulations using the crystal structures described in Chapter 2, which would help determine any differences to the dynamics of the desaturases and the hydroxylases. Additionally, MD simulations could help identify key residues that may be responsible for distinguishing their reactivity. Another way to identify these key residues would be to evolve the arginine oxidases in the lab to transform a desaturase into a hydroxylase, or vice versa. Studying the residues that alter the reactivity could provide clues to understand how this differentiation might have occurred naturally among the arginine oxidases. However, a major hurdle to this type of study is the development of an appropriate high-throughput assay that would be needed to distinguish the hydroxylated and desaturated products. Despite the progress in determining a mechanism for oxygen reactivity, the mechanistic details remain elusive, not only for the arginine oxidases, but for all oxygen-, PLP-dependent 141 enzymes. Future work in this area should consider using previous studies on flavin-dependent enzymes and cofactor-independent oxygenases as templates for exploring this reactivity.120,123,233 For example, researchers have done in-depth explorations of redox potentials to investigate oxygen reactivity of the flavin-dependent enzymes to support measured reaction rates and reveal the energetic influence of the enzyme on redox potential. With the arginine oxidases, measuring redox potentials of the enzyme and comparing with several mutagenic variants could help elucidate residues responsible for oxygen reactivity. Studies on flavin-dependent enzymes have also further investigated oxygen reactivity via more global comparisons between the oxygen-utilizing and non-utilizing examples.120,121 This analysis could also be applied to the arginine oxidases by comparing them with the most closely related PLP-dependent enzymes that do not use oxygen, which would first need to be analyzed phylogenetically to uncover the identity of these potential ancestors. Differences between oxygen usage in oxidases versus oxygenases has also been investigated in the flavin-dependent enzymes,120,121 which will serve as a useful template for a similar comparison amongst the oxygen- and PLP-dependent oxidases and oxygenases. The arginine oxidases could be useful biocatalysts in the future as they represent a unique and rare type of reaction amongst the highly versatile PLP-dependent enzymes. In order to be useful as a biocatalyst, it may be important for the oxygen-dependent hydroxylations and desaturations to be able to occur on a variety of different substrates. For this reason, the known arginine oxidases may need to be engineered to accept different substrates. Alternatively, once the basis for oxygen reactivity and the resulting hydroxylation and desaturation are known, the oxidase reactivities could be engineered into other PLP-dependent enzymes that accept other substrates. This type of engineering project would only be possible if it is confirmed that, as the data suggests, these reactivities are not dependent on the L-arginine substrate, itself, being involved in catalysis. Another potential use for the arginine oxidases is for the production of 2-ketoacids, which are attractive precursors in the production of many compounds because of their chemical versatility.234 Li et al. recently explored the use of aminotransferases for the production of 2-ketoacids by coupling aminotransferases with L-glutamate oxidase, catalase and a catalytic amount of L-glutamate to overcome issues with equilibrium and the need for an excess amount of amino acceptor.235 With mutation at a key active site His residue, the arginine oxidases are 142 also able to produce the 2-ketoacid, 2-ketoarginine, but without the need for a co-substrate amino acceptor or a co-substrate recycling enzyme. Engineering the arginine oxidases to accept a wider variety of substrates could enable them to be useful in a more efficient production of 2-ketoacids. 5.2.2 Indolmycin Further work with the indolmycin biosynthetic pathway and production of indolmycin-like compounds should involve understanding and improving the efficiency of production, the diversity of compounds and the bioactivity of indolmycin-like compounds. To improve the efficiency of production, more information should be gained about the pathway itself. In Chapter 4, when unnecessary genes were eliminated from the production of indolmycenic acid, the amount of indolmycenic acid produced was much less than for the system which included all of the genes from the initial attempt to make indolmycin biosynthetically. This finding indicated that the enzymes produced from these genes may in fact be necessary after all, if not catalytically, then perhaps through allosteric regulation. The role that some or all of these proteins may play on modulating the activity of Ind1 or Ind2 remains unclear but could hold key information needed to improve the efficiency of the system. Additionally, the heterologous expression system in E. coli produces a large amount of the shunt product, C-desmethyl-indolmycenic acid, which could be further optimized by understanding how regulation or environmental conditions within S. griseus ATCC 12648 could be mimicked in E. coli to improve flux through the desired indolmycenic acid intermediate. Gaining a better understanding of the regulation and optimal conditions for the indolmycin biosynthetic pathway may also enable purely biosynthetic production of indolmycin and its derivatives in the future. Improvements to the chemical synthesis would also result in a better yield for each compound from the semi-synthetic system. In order to improve the diversity of the indolmycin-like compounds, work should be done to expand the scope and optimize yield. The yield of indolmycin derivatives described in Chapter 4 could be improved by optimizing growth and isolation methods to increase the indolmycenic acid derivative yields. Another way to improve production of indolmycin derivatives through increasing indolmycenic acid derivative production would be to use a tryptophan auxotrophic strain of E. coli which would reduce the production of non-derivatized indolmycenic acid. To produce a greater diversity of indolmycin derivatives, first more indole 143 derivatives should be tested to better understand what is tolerated by the current system. Then, to improve the scope it will be necessary to understand why certain derivatives are not converted to indolmycenic acid derivatives. If the hurdle to conversion is uptake of the indole derivatives into E. coli, it would be useful to engineer a membrane transport system that could allow the indole derivatives to permeate the cell membrane. For indole derivatives whose conversion may be hindered by substrate acceptance from the biosynthetic enzymes, efforts could be placed into finding homologs with better substrate scopes or engineering the biosynthetic enzymes to improve their substrate scopes. Engineering TrpS for wider substrate conversion has already shown some success,83,90,228–232 but has not yet been attempted for Ind1 or Ind2, as crystal structures have not yet been solved for these enzymes. Regarding bioactivity, the antibiotic properties observed from the fluorinated indolmycin derivatives in Chapter 4 indicates that they may be useful in pharmaceutical development against MRSA. Since development of indolmycin was originally discontinued because of potential liver toxicity issues due to an effect on catabolic liver enzymes,34,36,37 it would be useful to test these fluorinated derivatives in vitro against these catabolic liver enzymes. It has also been suggested that C-desmethyl-indolmycin has a lower chance of causing hepatic toxicity while keeping some bioactivity against prokaryotic TrpRS,37 suggesting that it may be useful to optimize the semi-synthetic system to produce C-desmethyl-indolmycin derivatives to be tested for bioactivity against MRSA. Additionally, the indolmycin derivatives should also be tested against other pathogens shown to be targets of indolmycin, such as P. falciparum39 or H. pylori.32 Overall, this work has used the indolmycin biosynthetic pathway to help expand the known biocatalytic repertoire by gaining a better understanding of the oxygen- and PLP-dependent arginine oxidases and by producing new indolmycin derivatives semi-synthetically. By expanding the known biocatalytic repertoire, use of biocatalysts as chemical tools makes production of natural products and their derivatives more feasible. Industrial interest in natural products should increase as a result of greater accessibility to these complex molecules. 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Sci. 2001, 98 (18), 10037–10041. 166 Appendices Appendix A: Supplementary information for Chapter 3 UniProtKB ID Size Protein Family Predicted FunctionG8WNK6 367 PF00155 rohP; arginine hydroxylaseF8JPS0 169 N/A rohQ; guanidinoacetaldehyde cyclizationF8JVZ6 267 PF00701 rohR; retroaldolaseG8WNK3 289 PF14518 rohS; amino oxidaseF8JVZ5 95 PF00355 rohT; unknownUniProtKB ID Size Protein Family Predicted FunctionB4Y380 333 PF00303 milA; CMP 5-hydroxymethylaseB4Y381 169 PF05014 milB: CMP/hydroxymethyl CMP hydrolaseH9BDW2 419 N/A milC; cytosylglucuronic acid/hydroxymethyl cytosylglucuronic acid synthaseH9BDW3 394 PF01041 milD; degT/dnrJ/eryC1/strS aminotransferaseH9BDW4 272 PF01636 milE; aminoglycoside phosphotransferaseH9BDW5 156 N/A milF; hypothetical proteinH9BDW6 334 PF04055/PF13353 milG; radical SAM superfamilyH9BDW7 739 N/A milH; hypothetical proteinH9BDW8 359 PF00550 milI; chitinase AH9BDW9 316 N/A milJ; hypothetical proteinH9BDX0 441 PF07690 milK; major facilitator superfamilyH9BDX1 327 N/A milL; hypothetical proteinH9BDX2 388 PF00155 milM; aspartate/tyrosine/aromatic aminotransferaseH9BDX3 257 PF00701 milN; dihydrodipicolinate synthetase family proteinH9BDX4 355 PF00196 milO: regulatory protein LuxR familyH9BDX5 526 PF00005 milP: ABC transporter superfamilyH9BDX6 255 PF01636 milQ: aminoglycoside phosphotransferase1 kbmilA milB milC milD milE milF milG milH milI milJ milK milL milM milN milO milP milQ1 kbrohP rohQ rohR rohS rohTabAppendix Figure A-1: Example gene clusters with annotated gene functions for Group Ia. (a) Partial azomycin gene cluster from Streptomyces cattleya strain ATCC 35852 and table of annotated functions. (b) Streptomyces rimofaciens mildiomycin gene cluster and table of annotated functions. Sizes are given in number of amino acids. 167 UniProtKB ID Size Protein Family Predicted FunctionQ643C0 292 PF00155 mppP; PLP-dependent arginine hydroxylaseQ643B9 414 PF00155 mppQ; PLP-dependent aminotransferaseQ643B8 301 PF06314 mppR; dehydratase/cyclase1 kbmppP mppQ mppRaUniProtKB ID Size Protein Family Predicted FunctionA0A3R8QEI4 355 PF02826 dihydrofolate reductaseA0A3R8SFS3 391 N/A hypothetical proteinA0A3R8RHL4 474 PF03241/PF11794 4-hydroxyphenylacetate 3-monooxygenaseA0A426SBR1 317 PF08241 SAM-dependent methyltransferaseA0A426SC37 318 PF00491 agmatinaseA0A3R8QJY3 386 PF00155 hypothetical proteinA0A426SC80 371 PF04339 hypothetical proteinA0A3R8RFS1 339 PF00389/PF02826 phosphoglycerate dehydrogenase family proteinA0A426SBX5 404 PF00465 hypothetical proteinA0A3R8QDV1 452 N/A transcriptional regulatorA0A3R8RNY0 340 PF11583 hypothetical proteinA0A3R8S471 719 PF00733/PF13537 asparagine synthase (Glutamine-hydrolyzing)1 kbb168 UniProtKB ID Size Protein Family Predicted FunctionC7Q945 884 PF01804 peptidase S45 penicillin amidaseC7Q944 2249 PF00501/PF00550/PF00668 amino acid adenylation domain proteinC7Q943 389 PF00155 aminotransferaseC7Q942 342 PF02668 L-ornithine/L-arginine 3-hydroxylaseC7Q941 376 PF00155 aminotransferase class I and IIC7Q940 1338PF00501/PF00550/PF00668/PF00975/PF13193amino acid adenylation domain proteinC7Q939 192 PF01738 hypothetical proteinC7Q938 226 PF00196 Transcriptional regulator, LuxR familyC7Q937 379 PF00155 aminotransferase class I and IIc1 kbAppendix Figure A-2: Example gene clusters with annotated gene functions for Group II. (a) Streptomyces hygroscopicus enduracididine gene cluster and table of annotated functions. (b) Streptomyces griseofuscus unknown gene cluster and table of annotated functions. (c) Catenulispora acidiphila unknown gene cluster and table of annotated functions. Sizes are given in number of amino acids. 169 UniProtKB ID Size Protein Family Predicted FunctionA0A0D4BSA1 320 PF00579 ind0; tryptophanyl-tRNA synthaseA0A0D4BS77 328 PF13649 ind1; indolepyruvate C-methyltransferaseA0A0D4BSN8 569 N/A ind2; beta-methylindole-3-pyruvate reductaseA0A0D4BST1 451 N/A ind3; ATP-dependent amide synthetaseA0A0D4BS17 382 PF00155 ind4; PLP-dependent arginine desaturaseA0A0D4BSA6 318 PF00389/PF02826 ind5; NADPH-dependent imine reductaseA0A0D4BS80 228 N/A ind6; chaperone-likeA0A0D4BSP3 236 PF13649 ind7; N-demethylindolmycin N-methyltransferaseA0A0D4BST6 360 PF00155 ind8; aminotransferasea1 kbind0 ind1 ind2 ind3 ind4 ind5 ind6 ind7 ind8UniProtKB ID Size Protein Family Predicted FunctionA0A161S599 366 PF02875/PF08245 Mur ligaseA0A165QAT3 381 PF00155 pel4; aminotransferase class I and IIA0A163V3I0 318 PF00389/PF02826 pel5; imine/ketoacid reductaseA0A163V4B7 109 N/A protein tyrosine phosphataseA0A165QAS7 256 PF00455/PF08220 DeoR family transcriptional regulatorA0A163V3H0 440 PF01547 ABC transporter substrate-binding proteinA0A163V4A6 292 PF00528 ABC transporter permeaseA0A163V3F4 272 PF00528 sugar ABC transporter permeaseA0A165QAR7 583 PF00672 histidine kinaseA0A163V3E8 553 PF00072/PF12833 AraC family transcriptional regulatorA0A163V3D9 367 PF01168/PF14031 amino acid processing proteinA0A163V3C6 388 PF01546/PF07687 acetylornithine deacetylaseb1 kbpel4 pel5170 UniProtKB ID Size Protein Family Predicted FunctionA0A1I4FW50 390 PF00108/PF02803 acetyl-CoA acetyltransferaseA0A1I4FVH3 249 PF13304 predicted ATPaseA0A1I4FUF9 383 PF00155 aminotransferase class I and IIA0A1I4FUG4 305 PF00389/PF02826 bsp5; imine reductaseA0A1I4FVH2 455 N/A phenylacetate-CoA ligaseA0A1I4FXW7 291 PF08443 glutathione synthase/RimK-type ligase, ATP-grasp superfamilyA0A1I4FUJ5 406 PF13535 ATP-grasp domain-containing proteinA0A1I4FXS2 387 PF07690 predicted arabinose efflux permease, MFS familyc1 kbbsp5Appendix Figure A-3: Example gene clusters with annotated gene functions for Group Ib. (a) Streptomyces griseus indolmycin gene cluster and table of annotated functions. (b) Paenibacillus elgii unknown gene cluster and table of annotated functions. (c) Bacillus sp. 5mfcol3.1 unknown gene cluster and table of annotated functions. Sizes are given in number of amino acids. 171 UniProtKB ID Size Protein Family Predicted FunctionA0A167HIA4 380 PF00155 plu4; PLP-dependent arginine desaturaseA0A167HID2 315 PF00389/PF02826 plu5; 2-ketoacid dehydrogenaseA0A167HIE7 318 PF13847 plu1; indolepyruvate C-methyltransferaseA0A167HIE9 370 N/A plu2; beta-methylindole-3-pyruvate reductaseA0A167HIF2 441 N/A plu3; ATP-dependent amide synthetaseA0A167HIF8 304 N/A pluN1; unknownA0A161YDL9 236 PF13649 plu7; N-demethylindolmycin N-methyltransferaseA0A167HII1 131 PF00903 pluN2; chaperone-likeA0A167HIJ3 394 PF00155 plu8; aminotransferaseA0A167HIK5 332 PF00579 plu0; tryptophanyl-tRNA synthaseUniProtKB ID Size Protein Family Predicted FunctionA0A0D5XXF8 356 PF00389/PF02826 hydroxyacid dehydrogenaseA0A0D5XWM9 460 PF01565/PF08031 FAD-binding proteinA0A0D5XXQ4 459 PF01554 multidrug transporter MATEA0A0D5XWP5 607 N/A hypothetical proteinA0A0D5XXS7 389 PF00155 aspartate/tyrosine/aromatic aminotransferaseA0A0D5XXG3 526 PF09098/PF09099/PF09100/PF14930quinohemoprotein amine dehydrogenase, alpha subunitA0A0D5XWN3 476 PF04055/PF13353 quinohemoprotein amine dehydrogenaseA0A0D5XXQ9 106 PF08992 quinohemoprotein amine dehydrogenase, gamma subunitA0A0D5XWQ0 373 N/A quinohemoprotein amine dehydrogenase subunit beta PeaDA0A0D5XXT1 132 PF07007 urease-associated proteina1 kbplu4 plu5 plu1 plu2 plu3 pluN1 pluN2plu7 plu8 plu0b1 kb172 UnProtKB ID Size Protein Family Predicted FunctionA0A1H0X398 423 PF13535 ATP-grasp domain-containing proteinA0A1H0X1Y3 479 N/A phenylacetate-CoA ligaseA0A1H0X1U5 319 PF02746/PF13378 dipeptide epimeraseA0A1H0X1W9 445 PF00464 serine hydroxymethyltransferaseA0A1H0X1S5 389 PF00155 aspartate/methionine/tyrosine aminotransferaseA0A1H0X2G9 318 PF00389/PF02826 gluconate 2-dehydrogenaseA0A1H0X1T9 450 PF01565 FAD/FMN-containing dehydrogenaseA0A1H0X1R4 423 N/A hypothetical proteinA0A1H0X2G1 465 PF01554 Multidrug resistance protein, MATE familyA0A1H0X1T0 401 PF13535 biotin carboxylaseUniProtKB ID Size Protein Family Predicted FunctionA0A3M3S133 414 PF00464 putative serine hydroxymethyltransferaseA0A3M3MBM7 461 PF01554 putative multidrug resistance protein NorMA0A3M3MAJ3 475 N/A CapK domain proteinA0A3M3MAS6 315 PF00291 Pyridoxal-phosphate dependent enzyme proteinA0A3M3MA06 389 PF00155 aminotransferase class I and IIA0A3M3S0Z5 312 PF00389/PF02826 D-isomer specific 2-hydroxyacid dehydrogenase proteinA0A3M3S0C0 470 PF01565 putative oxidoreductase, FAD-bindingA0A3M3S095 530 PF01593 Amine oxidase, flavin-containing proteinA0A3M3S0Z6 409 PF02771/PF08028 acyl-CoA dehydrogenase proteinA0A3M3S0G4 394 PF02771/PF08028 acyl-CoA dehydrogenase proteinc1 kbd1 kb173 UniProtKB ID Size Protein Family Predicted FunctionA0A1K1Q0V2 395 PF00155 aminotransferase class I and IIA0A1K1Q0S4 310 PF00389/PF02826 lactate dehydrogenaseA0A1K1Q2L4 255 PF06182 ABC-type uncharacterized transport system, permease componentA0A1K1Q0V4 261 N/A ABC-2 type transport system permease proteinA0A1K1Q0A1 335 PF00005 ABC-2 type transport system ATP-binding proteinA0A1K1PZV1 486 PF00005 ATPase components of ABC transporters with duplicated ATPase domainsA0A1K1Q049 425 N/A phenylacetate-CoA ligaseUniProtKB ID Size Protein Family Predicted FunctionA0A2S9KP32 395 PF00389/PF02826 hypothetical proteinA0A2S9KP93 323 PF01564 polyamine aminopropyltransferaseA0A2S9KP81 357 N/A amidinotransferaseA0A2S9KP36 301 PF00005 ABC transporterA0A2S9KP34 267 PF06182 ABC transporter permeaseA0A2S9KP57 251 PF06182 membrane proteinA0A2S9KP38 447 PF00278/PF02784 putative diaminopimelate decarboxylaseA0A2S9KP24 407 PF00155 putative aspartate/tyrosine/aromatic aminotransferasee1 kbf1 kbAppendix Figure A-4: Example gene clusters with annotated gene functions for Group III. (a) Pseudoalteromonas luteoviolacea CPMOR-1 indolmycin gene cluster and table of annotated functions. (b) Pseudomonas chlororaphis unknown gene cluster and table of annotated functions. (c) Actinopolyspora xinjiangensis unknown gene cluster and table of annotated functions. (d) Pseudomonas syringae pv. Apii unknown gene cluster and table of annotated functions. (e) Pseudomonas sp. NFACC04-2 unknown gene cluster and table of annotated functions. (f) Burkholderia ambifaria unknown gene cluster and table of annotated functions. Sizes are given in number of amino acids. 174 Appendix B: Supplementary information for Chapter 4 0123456789101112131415f1 (ppm)- 500501001502002503003504004505005506006507007508008509002.971.201.010.991.000.960.970.971.006 .76.86.97.07.17.27.37.47.57.67.77.8f1 (ppm)01002003004000.991.000.960.970.971H NMRAppendix Figure B-1: 1H NMR (400 MHz, DMSO-d6) spectrum for indolmycenic acid (11). 175 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 5000500100015002000250030003500400045005000550060006500700075008000110115120125130135f1 (ppm)0200040006000800013C NMRAppendix Figure B-2: 13C NMR (101 MHz, DMSO-d6) spectrum for 11. 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f2 (ppm)0123456789101112 f1(ppm)6 .66.87.07.27.47.67.8f2 (ppm)6.57.07.58.0 f1(ppm)COSYNHOHO OHAppendix Figure B-3: COSY NMR (400 MHz, DMSO-d6), spectrum for 11. 176 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f2 (ppm)0102030405060708090100110120130140150160 f1(ppm)6 .86.97.07.17.27.37.47.57.67.7f2 (ppm)110115120125 f1(ppm)HSQCAppendix Figure B-4: HSQC NMR (400 MHz for 1H NMR, 101 MHz for 13C NMR, DMSO-d6) spectrum for 11. 177 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f2 (ppm)050100150200 f1(ppm)6 .87.07.27.47.67.8f2 (ppm)110120130140 f1(ppm)HMBC1.01.52.02.53.03.54 .04 .5f2 (ppm)110120130140 f1(ppm)NHOHO OHAppendix Figure B-5: HMBC NMR (400 MHz for 1H NMR, 101 MHz for 13C NMR, DMSO-d6) spectrum for 11. 178 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.5f1 (ppm)010020030040050060070080090010003.021.380.951.001.081.421.210.95024681012f1 (ppm)02004006003.021.380.951.001.081.421.210.951H NMRAppendix Figure B-6: 1H NMR (400 MHz, DMSO-d6) spectrum for 5-fluoro-indolmycenic acid (5F-11). - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)010002000300040005000600070008000900010000100105110115120125130135f1 (ppm)020004000600080001000013C NMRimpurityAppendix Figure B-7: 13C NMR (101 MHz, DMSO-d6) spectrum for 5F-11. 179 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.5f1 (ppm)- 2000200400600800100012001400160018002000220024002600280030003.321.190.920.901.811.010.846 .76 .86.97.07.17.27.37.47.57.6f1 (ppm)0500100015000.901.811.011H NMRAppendix Figure B-8: 1H NMR (400 MHz, DMSO-d6) spectrum for 6-fluoro-indolmycenic acid (6F-11). 180 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 5000050001000015000200002500030000350004000045000500005500060000100110120130140f1 (ppm)01000020000300004000013C NMRimpurityAppendix Figure B-9: 13C NMR (101 MHz, DMSO-d6) spectrum for 6F-11. 181 0123456789101112131415f1 (ppm)- 500501001502002503003504004505005503.121.050.981.950.990.940.976 .46 .56.66.76.86.97.07.17.27.37.47.57.67.77.8f1 (ppm)01002003004005001.950.990.941H NMRAppendix Figure B-10: 1H NMR (400 MHz, DMSO-d6) spectrum for 7-fluoro-indolmycenic acid (7F-11). 182 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 2000- 100001000200030004000500060007000800090001000011000120001300014000150001600017000180001900020000210002200023000105110115120125130135f1 (ppm)05000100001500013C NMRmethanolimpurityAppendix Figure B-11: 13C NMR (101 MHz, DMSO-d6) spectrum for 7F-11. 183 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f2 (ppm)0123456789101112 f1(ppm)6 .87.07.27.47.6f2 (ppm)6.57.07.5 f1(ppm)NHOHO OHFCOSYAppendix Figure B-12: COSY NMR (400 MHz, DMSO-d6) spectrum for 7F-11. 184 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f2 (ppm)0102030405060708090100110120130140150160 f1(ppm)6 .66 .87.07.27.47.6f2 (ppm)100110120130 f1(ppm)HSQCAppendix Figure B-13: HSQC NMR (400 MHz for 1H NMR, 101 MHz for 13C NMR, DMSO-d6) spectrum for 7F-11. 185 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f2 (ppm)- 20020406080100120140160180200 f1(ppm)6 .66.87.07.27.47.6f2 (ppm)100110120130140150 f1(ppm)HMBCNHOHO OHFAppendix Figure B-14: HMBC NMR (400 MHz for 1H NMR, 101 MHz for 13C NMR, DMSO-d6) spectrum for 7F-11. 186 0.51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.5f1 (ppm)0501001502002503003503.490.880.980.921.031.000.971.006 .97.07.17.27.37.47.57.67.7f1 (ppm)01002003000.921.031.000.971H NMRAppendix Figure B-15: 1H NMR (400 MHz, DMSO-d6) spectrum for 5-chloro-indolmycenic acid (5Cl-11). 187 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 2000200400600800100012001400160018002000220024002600280030003200340036003800105110115120125130135140f1 (ppm)010002000300013C NMRimpurityAppendix Figure B-16: 13C NMR (101 MHz, DMSO-d6) spectrum for 5Cl-11. 188 0.51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.513.0f1 (ppm)- 10001002003004005006007008009001000110012001300140015003.001.100.970.960.960.890.910.906 .97.07.17.27.37.47.57.6f1 (ppm)0200400600800100 00.960.960.890.911H NMRAppendix Figure B-17: 1H NMR (400 MHz, DMSO-d6) spectrum for 6-chloro-indolmycenic acid (6Cl-11). 189 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 500050010001500200025003000350040004500500055006000650070007500115120125f1 (ppm)020004000600013C NMRAppendix Figure B-18: 13C NMR (101 MHz, DMSO-d6) spectrum for 6Cl-11. -20. 190 - 0 .50 .00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.513.0f1 (ppm)- 40- 200204060801001201401601802002202402602803003203403603.023.501.001.010.930.960.960.926 .86.97.07.17.27.37.47.57.67.7f1 (ppm)01002003001.010.930.960.961H NMRAppendix Figure B-19: 1H NMR (400 MHz, DMSO-d6) spectrum for 7-chloro-indolmycenic acid (7Cl-11). 191 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 20002004006008001000120014001600180020002200240026002800110115120125130135f1 (ppm)01000200013C NMRAppendix Figure B-20: 13C NMR (101 MHz, DMSO-d6) spectrum for 7Cl-11. 192 - 0 .50 .00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.513.0f1 (ppm)- 200204060801001201401601802002202402603.052.760.960.941.011.061.010.981.020.870.881.01.11.21.31.4f1 (ppm)0501001502002503.052.62.72.82.9f1 (ppm)0501001502002502.763 .403.453.503.553.603.653.703.753.80f1 (ppm)02040600.964 .84 .95.05.1f1 (ppm)0501001502000.947.07.17.27.37.47.57.6f1 (ppm)0501001501.011.061.010.981.021H NMRAppendix Figure B-21: 1H NMR (400 MHz, DMSO-d6) spectrum for indolmycin (6). 193 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 1000010002000300040005000600070008000900010000110001200013000112114116118120122124126f1 (ppm)010002000300040005000600013C NMRmethanolimpurityAppendix Figure B-22: 13C NMR (101 MHz, DMSO-d6) spectrum for 6. 194 0.51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.5f2 (ppm)1234567891011f1 (ppm)7.07.5f2 (ppm)7.07.5f1 (ppm)10 .710 .810 .911.011.1f2 (ppm)6.97.07.17.27.37.4f1 (ppm)NHOONNHCOSYAppendix Figure B-23: COSY NMR (400 MHz, DMSO-d6) spectrum for 6. 195 0.51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.5f2 (ppm)0102030405060708090100110120130140150160f1 (ppm)6 .87.07.27.47.67.8f2 (ppm)105110115120125f1 (ppm)HSQCAppendix Figure B-24: HSQC NMR (400 MHz for 1H NMR, 101 MHz for 13C NMR, DMSO-d6) spectrum for 6. 196 0.51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.5f2 (ppm)050100150200f1 (ppm)7.07.5f2 (ppm)110120130140f1 (ppm)HMBCNHOONNHAppendix Figure B-25: HMBC NMR (400 MHz for 1H NMR, 101 MHz for 13C NMR, DMSO-d6) spectrum for 6. 197 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.513.0f1 (ppm)- 2002040608010012014016018020 02202402602803003203.172.981.000.991.143.460.990.986 .86.97.07.17.27.37.4f1 (ppm)0501001501.143.464 .754 .804.854.904.955.005.05f1 (ppm)0501001502000.993 .33.43.53.63.73.8f1 (ppm)- 200204060801001.002.62.72.82.9f1 (ppm)01002003002.981.01.11.21.31.4f1 (ppm)01002003.171H NMRAppendix Figure B-26: 1H NMR (400 MHz, DMSO-d6) spectrum for 5-fluoro-indolmycin (5F-6). 198 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 50005001000150020002500300035004000450050005500600065007000100105110115120125130135f1 (ppm)0100020003000400013C NMRAppendix Figure B-27: 13C NMR (101 MHz, DMSO-d6) spectrum for 5F-6. 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.5f2 (ppm)01234567891011f1 (ppm)6 .86.97.07.17.27.37.47.5f2 (ppm)6.57.07.5f1 (ppm)NHOONNHFCOSYAppendix Figure B-28: COSY NMR (400 MHz for 1H NMR, DMSO-d6) spectrum for 5F-6. 199 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f2 (ppm)102030405060708090100110120130f1 (ppm)6 .66.87.07.27.47.6f2 (ppm)100110120f1 (ppm)HSQCAppendix Figure B-29: HSQC NMR (400 MHz for 1H NMR, 101 MHz for 13C NMR, DMSO-d6) spectrum for 5F-6. 200 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.5f2 (ppm)102030405060708090100110120130140150160170180190f1 (ppm)6 .76.86.97.07.17.27.37.47.57.6f2 (ppm)100110120130f1 (ppm)NHOONNHFHMBCAppendix Figure B-30: HMBC NMR (400 MHz for 1H NMR, 101 MHz for 13C NMR, DMSO-d6) spectrum for 5F-6. 201 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f1 (ppm)12345677.98.08.18.28.38.48.58.68.78.88.9f1 (ppm)12345673.403.453.503.553.603.653.703.75f1 (ppm)12345674.74 .84 .95.05.1f1 (ppm)12345672.652.702.752.802.852.90f1 (ppm)12345670.91.01.11.21.31.41.51.6f1 (ppm)123456725 °C35 °C45 °C65 °C85 °C105 °C115 °C25 °C35 °C45 °C65 °C85 °C105 °C115 °C25 °C35 °C45 °C65 °C85 °C105 °C115 °C25 °C35 °C45 °C65 °C85 °C105 °C115 °C25 °C35 °C45 °C65 °C85 °C105 °C115 °C25 °C35 °C45 °C65 °C85 °C105 °C115 °CVaried Temperature NMRAppendix Figure B-31: Varied temperature NMR (400 MHz, DMSO-d6) spectra for 5F-6. 202 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.0f1 (ppm)- 200204060801001201401601802002202402602803003203403.473.001.010.961.052.131.011.030.996 .86.97.07.17.27.37.47.57.6f1 (ppm)0501001501.052.131.014 .804 .824.844.864.884.904.924.944.96f1 (ppm)0501001500.963 .403.453.503.553.603.653.70f1 (ppm)0204060801.012.62.72.82.9f1 (ppm)01002003003.001.01.11.21.3f1 (ppm)01002003003.471H NMRAppendix Figure B-32: 1H NMR (600 MHz, DMSO-d6) spectrum for 6-fluoro-indolmycin (6F-6). 203 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 2000200400600800100012001400160018002000220024002600280030003200100110120130f1 (ppm)0200400600800100013C NMRAppendix Figure B-33: 13C NMR (151 MHz, DMSO-d6) spectrum for 6F-6. 204 - 0 .50 .00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.513.0f1 (ppm)- 1001020304050607080901001101201301401501603.163.220.971.002.351.361.401.000.996 .86.97.07.17.27.37.47.5f1 (ppm)0204060801002.351.361.404 .804 .854.904.955.005.05f1 (ppm)0204060801001.000 .91.01.11.21.31.41.5f1 (ppm)0501001503.162.62.72.82.93.0f1 (ppm)0501001503.223 .43.53.63.73.8f1 (ppm)01020304050600.971H NMRAppendix Figure B-34: 1H NMR (600 MHz, DMSO-d6) spectrum for 7-fluoro-indolmycin (7F-6). 205 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 50005001000150020002500300035004000450050005500600065007000750080008500105110115120125130f1 (ppm)010002000300013C NMRAppendix Figure B-35: 13C NMR (151 MHz, DMSO-d6) spectrum for 7F-6. 206 0.51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.513.013.5f1 (ppm)0501001502002503003504004503.043.051.040.981.171.211.160.971.061.007.07.17.27.37.47.57.67.7f1 (ppm)01002001.171.211.160.974 .824 .844.864.884.904.924.944.964.985.00f1 (ppm)0501001502000.983 .453.503.553.603.653.70f1 (ppm)0204060801001.042.602.652.702.752.802.852.902.95f1 (ppm)01002003004003.051.01.11.21.31.4f1 (ppm)01002003003.041H NMRAppendix Figure B-36: 1H NMR (600 MHz, DMSO-d6) spectrum for 5-chloro-indolmycin (5Cl-6). 207 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 200- 10001002003004005006007008009001000110012001300140 015001600170018001900200 02100220 013C NMRAppendix Figure B-37: 13C NMR (151 MHz, DMSO-d6) spectrum for 5Cl-6. 208 0.00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.513.0f1 (ppm)- 2002040608010012014016018020 02202402602803003203403.003.000.851.051.431.421.361.360.930.906 .86.97.07.17.27.37.47.57.67.7f1 (ppm)01002003001.431.421.361.363 .43.53.63.7f1 (ppm)0501001502000.854 .754 .804.854.904.955.005.05f1 (ppm)0501001501.052.62.72.82.9f1 (ppm)01002003003.001.01.11.21.31.4f1 (ppm)01002003003.001H NMRAppendix Figure B-38: 1H NMR (400 MHz, DMSO-d6) spectrum for 6-chloro-indolmycin (6Cl-6). 209 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 500050010001500200 02500300 035004000450050005500600065007000750013C NMRAppendix Figure B-39: 13C NMR (101 MHz, DMSO-d6) spectrum for 6Cl-6. 210 - 0 .50 .00 .51.01.52.02.53.03.54 .04 .55.05.56.06.57.07.58.08.59.09.510 .010 .511.011.512.012.513.0f1 (ppm)- 1001020304050607080901001101201301401502.903.001.021.001.232.291.230.891.056 .97.07.17.27.37.47.57.67.7f1 (ppm)0501001.232.291.234 .74 .84 .95.05.1f1 (ppm)- 200204060801001201.003 .43.53.63.7f1 (ppm)02040601.022.602.652.702.752.802.852.902.95f1 (ppm)0501003.001.01.11.21.31.41.5f1 (ppm)0501002.901H NMRAppendix Figure B-40: 1H NMR (400 MHz, DMSO-d6) spectrum for 7-chloro-indolmycin (7Cl-6). 211 - 20- 100102030405060708090100110120130140150160170180190200210220f1 (ppm)- 500050010001500200 02500300 03500400045005000550060006500700013C NMRAppendix Figure B-41: 13C NMR (101 MHz, DMSO-d6) spectrum for 7Cl-6.
Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Building chemical tools from the indolmycin biosynthetic pathway
Text