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The catabolism of the last two rings of cholesterol by Mycobacterium tuberculosis Crowe, Adam Michael 2018

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  THE CATABOLISM OF THE LAST TWO RINGS OF CHOLESTEROL BY MYCOBACTERIUM TUBERCULOSIS by  Adam Michael Crowe  B.Sc., The University of British Columbia, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   February 2018  © Adam Michael Crowe, 2018  ii  Abstract Cholesterol, a four-ringed steroid with an alkyl side chain, is an important growth substrate for Mycobacterium tuberculosis (Mtb) during infection. Many aspects of this catabolism remain unknown although steroid catabolism is a defining feature of mycobacteria and the related rhodococci. Using a variety of approaches, I elucidated key aspects of cholesterol catabolism in Mtb and other bacteria, particularly with respect to 3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indane-dione (HIP), a metabolite that contains the last two steroid rings (C/D). Chapter 2 demonstrates that the first two steroid rings (A/B) are degraded prior to the side chain in mycobacteria and rhodococci. This was established by targeting HsaD, the final ings A/B-degrading enzyme. Thus, a ΔhsaD mutant of Rhodococcus jostii RHA1 accumulated cholesterol-derived catabolites with partially degraded side-chains. Moreover, HsaD from Mtb had 100-fold higher specificity (kcat/KM) for a metabolite with a partially-degraded side chain. Chapter 3 presents a mechanism for KstR2, a TetR family transcriptional repressor that regulates the HIP catabolic genes, including ipdABCF and echA20. The KstR2 dimer bound two equivalents of HIP-CoA with high affinity (KD = 80±10 nM). Crystallographic analyses revealed that HIP-CoA binding induces conformational changes in the dimer that preclude DNA-binding. Mutagenesis substantiated the roles of Arg162 and Trp166 in HIP-CoA binding. In Chapter 4, key HIP catabolic steps are elucidated. Two previously undescribed metabolites, 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH-HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) were identified using deletion mutants of ipdC and ipdAB, respectively, combined with novel metabolomics approaches. Together, purified IpdC, IpdF and EchA20 transformed 5-OH-HIC-CoA to COCHEA-CoA. These data, along with those from additional mutants, were used to formulate a iii  HIP catabolic pathway and to predict that cholesterol catabolism yields four propionyl-CoA, four acetyl-CoA, one pyruvate, and one succinate. Chapter 5 establishes that IpdAB catalyzes a retro-Claisen-like ring-opening of COCHEA-CoA (kcat/KM = 2±0.7 × 105 M-1s-1) despite structural similarity with Class I CoA transferases. Based on crystal structures of IpdAB and biochemical data, a mechanism for ring-cleavage is proposed in which conserved Glu105 acts as a catalytic base. Overall, this work significantly advances our understanding of bacterial steroid catabolism and facilitates the development of novel therapeutics to treat TB.   iv  Lay Summary The ability of Mycobacterium tuberculosis (Mtb) to cause tuberculosis depends on the pathogen’s ability to degrade host-derived cholesterol, a 4-ringed steroid with a side chain. I used a variety of techniques to provide novel insights into the degradation of cholesterol in Mtb and related bacteria. Firstly, I demonstrated that the first two cholesterol rings are degraded prior to the side chain in Mtb and related bacteria. Secondly, I described how a two-ringed cholesterol metabolite regulates cholesterol degradation genes. Thirdly, I elucidated the pathway by which the last two cholesterol rings are degraded in Mtb and related bacteria. Finally, I established that IpdAB, a virulence factor in Mtb, cleaves open the last cholesterol ring and proposed a mechanism for how the enzyme does this. This dissertation provides insight into bacterial steroid degradation and facilitates the development of novel therapeutics to treat TB. v  Preface Chapter 1: Figure 1.5 and Figure 1.6 were adapted from Capyk et al. (2012) and Griffin et al. (2012), respectively, with permission from appropriate sources. Portions of the introductory text are used with permission from Crowe et al. (2015) of which I am first author. I created Table 1.1, modifying it to summarize data from Capyk et al. (2012), Griffin et al. (2012), Nesbitt et al. (2010), and Pandey et al. (2008).  Chapter 2: The contents of this chapter were prepared for submission in 2018 as: ‘Crowe, A., Brown, K., Casabon, I., Kulkarani, J., Yam, K., and Eltis, L.D. The unusual convergence of steroid catabolic pathways in Mycobacterium abscessus’. Sections, 2.2.4, and 2.3.3 were performed and written by KB. NMR structure elucidation of DHSBNC was performed by KY. Production of CoA thioesterified substrates was aided by IC. All other sections were performed by myself and written by LE and myself. All work presented in Chapter 2 was performed at The University of British Columbia, Vancouver, Canada. Chapter 3: A version of this material has been published as: ‘Crowe, A., Stogios, P., Casabon, I., Evdokimova, E., Savchenko, A., and Eltis, L.D. (2015). Structural and functional characterization of a ketosteroid transcriptional regulator of Mycobacterium tuberculosis. Journal of Biological Chemistry. 290(2): 872-882’. Experiments relating to the crystallization of KstR2 and structure refinement were performed by PS and EE at the University of Toronto, Toronto, Canada. HIP-CoA for crystallography was produced by IC. Sections 3.2.5 and 3.3.2 were performed and written by PS and EE. All other experiments were performed by myself and all other sections were written by LE and myself. Chapter 4: A version of this material has been published as: ‘Crowe, A., Casabon, I., Brown, K., Liu, J., Lian, J., Rogalski, J., Hurst, T., Snieckus, V., and Eltis, L.D. (2017). vi  Catabolism of the last two steroid rings in Mycobacterium tuberculosis and other bacteria. mBio 8(2) 00321-17’. Design and optimization of extraction of CoA thioester metabolites and much of the LC-MS analysis were performed by IC with help from JR (The University of British Columbia, Vancouver, Canada). Gene deletion and growth of Mtb was performed by KB and JL (The University of British Columbia, Vancouver, Canada). Macrophage infection studies were performed by KB. 5α-OH-HIC and 5β-OH-HIC were chemically synthesized by TH and VS at Queen’s University, Kingston, Canada. All other experiments were performed by myself. Section 4.2.4 was written by KB. All other sections were written by LE and myself. Chapter 5: The contents of this chapter were prepared for submission in 2018 as: ‘Crowe, A., Workman, S., Worrall, L., Watanabe, N., Casabon, I, Strynadka, N., and Eltis L.D. IpdAB, a virulence factor in Mycobacterium tuberculosis, is a cholesterol ring-cleaving hydrolase’. Experiments and figures relating to the refinement of X-ray crystal structures were performed by SW, LW, and NW at The University of British Columbia, Vancouver, Canada. Intact protein mass spectrometry was performed at The Center for High-Throughput Biology, The University of British Columbia. SW, LW, and NS were the primary authors of Sections 5.2.7 and 5.2.8 and made significant contributions to the writing and analysis described in Sections 5.3.4, 5.3.5, 5.3.6, and 6.4.1. All other work was performed by myself and all other sections were written by LE and myself. Chapter 6: This material was adapted from the publications listed above for Chapters 2-5. All sections were written by LE and myself. vii  Table of Contents  Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................xv List of Figures ............................................................................................................................. xvi List of Abbreviations ................................................................................................................. xix Acknowledgements .................................................................................................................... xxi  Introduction ..................................................................................................................1 Chapter 1:1.1  Steroids ........................................................................................................................... 1 1.1.1  Cholesterol .................................................................................................................. 2 1.2  Bacterial metabolism of steroids ..................................................................................... 5 1.2.1  Cholesterol catabolic pathway .................................................................................... 8 1.2.2  Cholesterol import .................................................................................................... 10 1.2.3  Side chain degradation .............................................................................................. 11 1.2.4  Cholesterol Rings A and B degradation .................................................................... 14 1.2.5  Cholesterol Rings C and D degradation .................................................................... 16 1.2.6  Concurrent side chain and Rings A and B degradation ............................................ 17 1.2.7  Organization and regulation of steroid catabolic gene clusters ................................ 17 1.2.8  Cholate and testosterone catabolism ......................................................................... 21 1.3  Mycobacterium tuberculosis and tuberculosis .............................................................. 21 viii  1.3.1  Mycolata ................................................................................................................... 23 1.3.1.1  Mycobacterium abscessus ................................................................................. 24 1.3.1.2  Mycobacterium smegmatis ................................................................................ 24 1.3.1.3  Rhodococcus jostii RHA1 ................................................................................. 24 1.3.2  Lipid metabolism in Mtb ........................................................................................... 25 1.3.3  Role of cholesterol catabolism in Mtb pathogenesis ................................................. 27 1.3.3.1  Cholesterol catabolism as a target for novel therapeutics ................................. 29 1.4  Coenzyme A (CoA) ...................................................................................................... 30 1.5  Meta-cleavage product (MCP) hydrolases .................................................................... 32 1.5.1  Structure of MCP hydrolases .................................................................................... 33 1.5.2  Mechanism of serine-dependent MCP hydrolases .................................................... 33 1.6  TetR family of transcriptional repressors ..................................................................... 35 1.7  Coenzyme A transferases .............................................................................................. 36 1.7.1  The ipdAB genes encode a predicted CoA transferase ............................................. 36 1.7.2  Classes of CoA transferases ...................................................................................... 37 1.7.3  Structure of Class I Coenzyme A transferases .......................................................... 37 1.7.4  Mechanism of Class I Coenzyme A transferases ...................................................... 38 1.8  Aim of this study ........................................................................................................... 39  Steroid catabolism in M. abscessus provides insights into the order of cholesterol Chapter 2:Rings A and B and sidechain degradation in actinobacteria ...................................................42 2.1  Introduction ................................................................................................................... 42 2.2  Materials and methods .................................................................................................. 43 2.2.1  Chemicals and reagents ............................................................................................. 43 ix  2.2.2  DNA manipulation .................................................................................................... 43 2.2.3  Phylogenetic analyses ............................................................................................... 44 2.2.4  Growth of bacterial strains ........................................................................................ 44 2.2.5  Metabolite analysis ................................................................................................... 45 2.2.6  Purification of HsaCMtb, HsaDMtb, HsaDMtb S114A, and HsaDMab ........................... 45 2.2.7  Preparation of substrates ........................................................................................... 46 2.2.8  Steady- state kinetic analyses.................................................................................... 47 2.2.9  KD determination ....................................................................................................... 48 2.3  Results ........................................................................................................................... 48 2.3.1  Bioinformatic analysis of the steroid catabolic gene cluster in M. abscessus .......... 48 2.3.2  M. abscessus possesses a HsaD ortholog dissimilar from other actinobacteria ....... 52 2.3.3  M. abscessus grows on cholesterol and 4-AD but not cholate ................................. 55 2.3.4  ΔhsaC and ΔhsaD RHA1 accumulate metabolites with incompletely degraded sidechains. ............................................................................................................................. 56 2.3.5  The substrate specificities of HsaCMtb, HsaDMab and HsaDMtb. ................................. 58 2.3.5.1  HsaDMtb has high affinity for substrates with partially degraded side chains ... 60  Structural and functional characterization of the KstR2∙HIP-CoA complex ......63 Chapter 3:3.1  Introduction. .................................................................................................................. 63 3.2  Materials and methods .................................................................................................. 64 3.2.1  Chemicals and reagents ............................................................................................. 64 3.2.1.1  Preparation of HIP and HIP-CoA ..................................................................... 64 3.2.2  DNA manipulation .................................................................................................... 65 3.2.3  Purification of KstR2 and variants ............................................................................ 65 x  3.2.4  Functional characterization of KstR2 ....................................................................... 66 3.2.4.1  Isothermal titration calorimetry ........................................................................ 66 3.2.4.2  Electrophoretic mobility shift assays ................................................................ 66 3.2.4.3  Size exclusion chromatography ........................................................................ 67 3.2.5  Crystallization of KstR2Mtb:HIP-CoA....................................................................... 68 3.2.5.1  Structural analysis ............................................................................................. 68 3.3  Results ........................................................................................................................... 69 3.3.1  KstR2Mtb binds HIP-CoA with high affinity ............................................................. 69 3.3.2  Structure of KstR2Mtb:HIP-CoA reveals an effector binding cleft spanning the two protomers of the dimer .......................................................................................................... 70 3.3.3  Binding of HIP-CoA alters the conformation of KstR2 ........................................... 73 3.3.4  Functional validation of KstR2:HIP-CoA interactions ............................................. 77 3.3.5  A KstR2 operator sequence binds two KstR2 dimers ............................................... 79  Elucidation of HIP catabolism in M. tuberculosis and actinobacteria ..................81 Chapter 4:4.1  Introduction ................................................................................................................... 81 4.2  Materials and methods .................................................................................................. 82 4.2.1  Chemicals and reagents ............................................................................................. 82 4.2.1.1  Preparation of steroid metabolites and CoA thioesters ..................................... 82 4.2.1.1.1  Purification of COCHEA-CoA ................................................................... 83 4.2.1.1.2  Purification of MOODA .............................................................................. 84 4.2.2  DNA manipulation, plasmid construction, and gene deletions................................. 84 4.2.3  Growth of bacteria .................................................................................................... 85 4.2.4  Macrophage infections .............................................................................................. 86 xi  4.2.5  Preparation of CoA metabolomes ............................................................................. 86 4.2.6  CoA metabolite profiling by LC-MS ........................................................................ 87 4.2.6.1  Targeted LC-MS ............................................................................................... 87 4.2.6.2  Untargeted LC-MS ............................................................................................ 88 4.2.6.3  NMR characterization of metabolites ............................................................... 88 4.2.6.4  Analysis of CoA metabolomics data ................................................................. 89 4.2.7  Protein production and purification .......................................................................... 89 4.2.8  Enzymatic transformations ....................................................................................... 91 4.2.9  Bioinformatic analyses.............................................................................................. 92 4.3  Results ........................................................................................................................... 92 4.3.1  The ipdABC genes are required for growth on cholesterol and HIP ......................... 92 4.3.2  Growth in macrophages ............................................................................................ 95 4.3.3  The accumulation of cholesterol catabolites in the ipd mutants ............................... 95 4.3.4  Identification of CoA metabolites in the ipd mutants ............................................. 100 4.3.5  Enzymatic transformation of 5-OH-HIC-CoA ....................................................... 101 4.3.6  Bioinformatic analysis of HIP catabolic enzymes .................................................. 105 4.3.7  Validation of HIP catabolism using additional mutants ......................................... 107 4.3.8  HIP-dependent toxicity ........................................................................................... 110  Structural and mechanistic characterization of IpdAB .......................................113 Chapter 5:5.1  Introduction ................................................................................................................. 113 5.2  Materials and methods ................................................................................................ 114 5.2.1  Chemicals and reagents ........................................................................................... 114 5.2.2  Bioinformatic anaylsis of CoA transferases ........................................................... 114 xii  5.2.3  DNA manipulation and plasmid construction: ....................................................... 114 5.2.4  Production of COCHEA-CoA: ............................................................................... 115 5.2.5  Protein production and purification: ....................................................................... 116 5.2.6  Characterization of IpdAB and variants: ................................................................ 116 5.2.7  Crystallization of IpdAB and variant ...................................................................... 117 5.2.8  Crystallographic analysis and refinement: .............................................................. 117 5.2.9  In vitro activity of IpdAB ....................................................................................... 118 5.2.9.1  Steady-state kinetic characterization of IpdAB: ............................................. 118 5.2.10  Structure assignment for MOODA-CoA: ........................................................... 119 5.2.11  Attempts to trap acyl-enzyme intermediates ....................................................... 119 5.2.12  KD determination for IpdAB E105AA: ................................................................ 120 5.2.13  Deuterium incorporation into COCHEA-CoA: .................................................. 120 5.2.14  Proton-deuterium exchange NMR experiments: ................................................ 121 5.2.15  18O labelling of COCHEA-CoA, MOODA-CoA, and IpdAB ............................ 121 5.2.16  Mass Spectrometry: ............................................................................................ 122 5.3  Results ......................................................................................................................... 123 5.3.1  Phylogenetic analysis of IpdAB and Class I and II CoA transferases .................... 123 5.3.2  Characterization of IpdAB ...................................................................................... 124 5.3.2.1  In vitro transformations and steady state kinetics ........................................... 125 5.3.3  IpdAB catalyzed the efficient transformation of COCHEA-CoA .......................... 126 5.3.4  The structural fold of IpdAB is typical of Class I CoTs ......................................... 128 5.3.5  IpdAB has distinct active site residues ................................................................... 129 5.3.6  Structure of IpdAB·COCHEA-CoA complexes ..................................................... 131 xiii  5.3.7  Identification of catalytically essential residues ..................................................... 133 5.3.8  The IpdAB reaction mechanism does not appear to involve a glutamyl-CoA intermediate ......................................................................................................................... 135 5.3.9  Formation of a β-keto enolate in the E105AA variant ............................................. 136 5.3.10  IpdAB catalyzed deuteration of COCHEA-CoA ................................................ 137 5.3.11  18O is not incorporated into COCHEA-CoA or IpdAB ...................................... 140  Discussion ..................................................................................................................142 Chapter 6:6.1  Cholesterol Rings A and B are degraded prior to the alkyl side chain ....................... 142 6.1.1  Steroid degradation pathways in Mab appear to share a single HsaD .................... 143 6.1.2  HsaDMtb and HsaDMab have highest specificity for steroid substrates with incompletely degraded side chains ..................................................................................... 144 6.1.3  Updates to the pathway of cholesterol degradation ................................................ 146 6.2  HIP-CoA binding to KstR2 regulates catabolism of cholesterol Rings C and D ....... 147 6.2.1  Comparisons to TetR family of transcriptional repressors ..................................... 148 6.2.2  Insights into the inducer of KstR ............................................................................ 151 6.3  Elucidation of the catabolism of cholesterol Rings C and D ...................................... 152 6.3.1  The catabolic pathway of cholesterol Rings C and D in actinobacteria ................. 152 6.4  IpdAB is a ring-cleaving hydrolase ............................................................................ 154 6.4.1  Proposed mechanism of IpdAB .............................................................................. 155 6.5  Broader implications from the elucidation of HIP catabolism ................................... 156 6.5.1  The HIP catabolic pathway in other bacteria .......................................................... 156 6.5.2  Cleavage of MeDODA-CoA likely requires an unidentified thiolase .................... 158 6.5.3  HIP catabolism displays similarities with anaerobic aromatic catabolism ............. 158 xiv  6.6  Insights for Mtb pathogenicity and therapeutic development ..................................... 159 6.6.1  Disruption of KstR2 genes yield a ‘cholesterol-dependent-toxicity’ in Mtb .......... 159 6.6.2  Therapeutics targeting Mab .................................................................................... 160 6.6.3  Therapeutics targeting IpdAB ................................................................................. 161 6.7  Remaining questions and future directions ................................................................. 162 6.7.1  Cholesterol side chain and Rings A and B degradation .......................................... 162 6.7.2  Regulation of cholesterol catabolism ...................................................................... 162 6.7.3  Elucidation of HIP catabolism ................................................................................ 163 6.7.4  Characterization of IpdAB ...................................................................................... 164 Bibliography ...............................................................................................................................165 Appendices ..................................................................................................................................181 Appendix A Bacterial strains, plasmids and oligonucleotides in Chapters 4 and 5 ............... 181 Appendix B X-ray crystallography data collection and statistics ........................................... 185 B.1  KstR2: HIP-CoA crystallography statistics ............................................................ 185 B.2  IpdAB, IpdAB: COCHEA-CoA, and IpdAB E105AA: COCHEA-CoA crystallography statistics ..................................................................................................... 186 Appendix C Analyses of metabolites ...................................................................................... 187  xv  List of Tables   Table 1.1. Cholesterol catabolic genes in Mtb ................................................................................ 9 Table 2.1 The steroid and monoaromatic catabolic genes in M. abscessus ATCC 19977 ........... 51 Table 2.2 Steady-state kinetic parameters of HsaCMtb, HsaDMtb and HsaDMtb ............................. 60 Table 2.3 Parameters for dissociation constant determination of HsaDMtb .................................. 62 Table 3.1 Thermodynamic parameters of KstR2Mtb binding HIP-CoA. ....................................... 69 Table 4.1 Characterization of CoA metabolites found in this study ............................................. 97 Table 4.2 Annotation of KstR2 regulon ...................................................................................... 105 Table 5.1 Steady state parameters for FadA6 and IpdAB .......................................................... 126 Table 5.2 Specific activity of IpdAB variants ............................................................................ 134  xvi  List of Figures   Figure 1.1. Chemical structures of representative steroids. ............................................................ 2 Figure 1.2. The cholesterol biosynthetic pathway in animals. ........................................................ 4 Figure 1.3. Evolutionary relationship between steroid degrading bacteria. ................................... 6 Figure 1.4 Cholesterol catabolic pathway in Mtb. ........................................................................ 12 Figure 1.5. Organization of the cholesterol catabolic gene cluster in Mtb. .................................. 19 Figure 1.6. Central metabolic pathways of Mtb involved in fatty acid and cholesterol catabolism........................................................................................................................................................ 26 Figure 1.7 Structure of Coenzyme-A. ........................................................................................... 30 Figure 1.8 Mechanism of HsaD and MCP hydrolases. ................................................................. 35 Figure 1.9 Mechanism of CoA Transferases. ............................................................................... 39 Figure 2.1 The steroid catabolic gene clusters in M. abscessus .................................................... 54 Figure 2.2 Growth of M. abscessus on different steroids. ............................................................ 56 Figure 2.3 ΔhsaC and ΔhsaD RHA1 accumulate cholesterol derived metabolites with partially degraded side chains. .................................................................................................................... 58 Figure 2.4 Steady-state kinetic parameters of HsaCMtb and MCP hydrolases towards substrates with and without partially degraded side chains ........................................................................... 59 Figure 2.5 Dissociation constant (Kd) determination for HsaDMtb towards DSHBNC-CoA. ....... 61 Figure 3.1 Representative isotherms of potential KstR2Mtb ligands. ............................................ 70 Figure 3.2 Crystal structure of KstR2Mtb·HIP-CoA complex. ...................................................... 73 Figure 3.3 Conformational differences between KstR2Mtb·HIP-CoA and ligand-free KstR2RHA1 76 xvii  Figure 3.4 . Comparison of KstR2 structures with a TFR·DNA complex ................................... 77 Figure 3.5 Isotherms of KstR2Mtb variants. ................................................................................... 78 Figure 3.6 EMSA of KstRMtb and variants.................................................................................... 79 Figure 3.7 SEC-MALS of KstR2Mtb ............................................................................................. 80 Figure 4.1 Growth of ΔipdAB Mtb................................................................................................ 92 Figure 4.2 Growth and CoA metabolites of RHA1 strains ........................................................... 94 Figure 4.3 Growth and CoA metabolites of ΔipdC Mtb. .............................................................. 95 Figure 4.4 Accumulation of cholesterol derived metabolites from ΔipdAB and ΔipdC strains. .. 99 Figure 4.5 Electrophoretic analyses of gene deletion mutants and purified proteins. ................ 102 Figure 4.6 LC/MS analyses of the transformation of 5-OH HIC-CoA by purified enzymes. .... 104 Figure 4.7 Cholesterol-derived metabolite of ΔfadE32 M.smegmatis. ....................................... 107 Figure 4.8 Characterization of KstR2 regulon mutants of M. smegmatis. .................................. 109 Figure 4.9 Metabolites produced by M. smegmatis strains. ........................................................ 110 Figure 4.10 Cholesterol-dependent toxicity. ............................................................................... 112 Figure 5.1 Bioinformatic analysis of IpdAB and homologs. ...................................................... 123 Figure 5.2 In vitro activity of IpdAB. ......................................................................................... 125 Figure 5.3 Structure of IpdABRHA1 ............................................................................................. 128 Figure 5.4 Structure of IpdABCOCHEA-CoA. ......................................................................... 130 Figure 5.5 Characterization of IpdAB ........................................................................................ 134 Figure 5.6 IpdAB is not inhibited by sodium borohydride. ........................................................ 135 Figure 5.7 IpdAB E105AA stabilizes a yellow coloured species. ............................................... 136 Figure 5.8 IpdAB catalyzes proton exchange on COCHEA-CoA .............................................. 138 Figure 5.9 NMR characterization of IpdAB catalyzed deuterium incorporation ....................... 139 xviii  Figure 5.10 Additional NaBH4 and 18O experimental data ......................................................... 140 Figure 6.1 Proposed pathway of cholesterol catabolism in actinobacteria. ................................ 146 Figure 6.2 Proposed HIP catabolic pathway. .............................................................................. 153 Figure 6.3 Proposed mechanism of IpdAB. ................................................................................ 155  xix  List of Abbreviations 4-AD 4-androstene-3,17-dione 5OH-HIC 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone ADD 1,4-androstadiene-3,17-dione BLAST Basic Local Alignment Search Tool CoA Coenzyme A COCHEA-CoA (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA DHSA 3,4-Dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione DHSBNC 3,4-dihydroxy- 17-isopropionoyl- 9,10-seconandrost-1,3,5(10)-triene-9-one DSHA 4,5–9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid DSHBNC 4,5-9,10-diseco-3-hydroxy-5,9-dioxo-23,24-bisnorchola-1(10),2-dien-4,22-dioate FMN Flavin Mononucleotide FPLC Fast Protein Liquid Chromatography GC-MS Gas Chromatography coupled Mass Spectrometry HIP 3aα-H-4α(3’-propanoate)-7aβ-methylhexahydro-1,5-indanedione HPLC High Performance Liquid Chromatography IPTG Isopropyl β-D-1-thiogalactopyranoside LB Lysogeny Broth LC-MS Liquid Chromatography coupled Mass Spectrometry M9G M9 basal salts with Ca/Mg/Thiamine and Goodies Mix Media MOODA 4-methyl-5-oxo-octanedioc acid Mtb Mycobacterium tuberculosis Mab Mycobacterium abscessus NAD+ Nicotinamide adenine dinucleotide (oxidized) xx  NADH Nicotinamide Adenine Dinucleotide (reduced) NCBI National Center for Biotechnology Information NMR Nuclear Magnetic Resonance OD600 Optical density at 600 nm PCR Polymerase Chain Reaction PDB Protein Data Bank r.m.s.d Root Mean Square Distance RHA1 Rhodococcus jostii RHA1 SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis      xxi  Acknowledgements I would like to thank my supervisor, Prof. Lindsay D. Eltis for his patience and insights into the work presented herein. I am ingratiated for the research opportunities and learning experiences that you have provided me with.     Both my committee members, Prof. Natalie Strynadka and Prof. Martin Tanner have been an invaluable source of wisdom throughout my research.   I would like to thank all of the current and previous members of the Eltis Lab: Dr. Jenna Capyk, Dr. Israel Casabon, Dr. Nicolas Seghezzi, Dr. Hiroshi Otani, Dr. Rahul Singh, Dr. Antonio Ruzzini, Dr. Morgan Fetherolf, Jonathan Penfield, Carlos Diaz-Salazar, Eugene Kuatsjah, Keith Story, Jie Liu, and Jennifer Lian, for their expertise and advice. I would like to especially thank Kirstin Brown, James Round, and Raphael Roccor for their insights and friendship throughout my years in the Eltis Lab.   I have been lucky to collaborate with many individuals. I would like to thank Dr. Leonard Foster (UBC), Dr. William Mohn (UBC), Dr. Victor Snieckus (Queen’s University, Kingston, ON), Dr. Alexei Savchenko (The University of Toronto, ON), Dr. Timothy Hurst (Queen’s University), Dr. Peter Stogios (The University of Toronto), Dr. Liam Worrall (UBC), Dr. Nobu Watanabe, Dr. Sean Lott (University of Auckland, Auckland, New Zealand), and Sean Workman (UBC). I would especially like to thank Mark Okon (UBC) and Jason Rogalski (UBC) for their aid with NMR and LC-MS, respectively.  I would like to acknowledge the financial support from the Canadian Institute of Health Research (CIHR), The BC Lung Association, and The University of British Columbia.  xxii   Lastly, I would like to thank my loving wife, Amanda Deacon, for her patience over the years. Her continued support has eased the trials and tribulations of my PhD. I could not have succeeded without you.    1   Introduction Chapter 1:1.1 Steroids Steroids are polycyclic organic molecules with diverse functions across eukaryotes and prokaryotes [1]. The core steroid structure consists of three cyclohexane rings (Rings A, B, and C) and a single cyclopentane ring (Ring D). As exemplified in Figure 1.1, this core domain can be modified to include a branched alkyl side chain at carbon 17 (C-17), as with cholesterol, β-sitosterol, and lanosterol; a hydroxylated alkyl side chain at C-17, as with cholic acid and progesterones; or only hydroxylation at C-17, as with the androgens [1]. Similarly, the cycloalkyl rings can display multiple hydroxylations, as with cholic acids; a single hydroxylation at C-3, as with cholesterol, lanosterol, and β-sitosterol; or a C-3 oxo, as with the androgens and progesterones [1].  Additional chemical modifications include aromatization of Ring A, as with the estrogens, and desaturations, as found in many steroids [1].  Animals, plants, and fungi use steroids for a number of vital functions. In animals, cholesterol can be enzymatically modified to yield a broad range of steroid hormones. In humans, these include the sex hormones, testosterone, estradiol and progesterone, as well as the mineralocorticoids and glucocorticoids. Bile acids, derived from cholic acid, are utilized in the emulsification of fats within the small intestine. Across all animals, cholesterol is employed to maintain membrane fluidity. In fungi, membrane fluidity is maintained by ergosterol [2]. Plant steroids, referred to as phytosterols, similarly act as signaling molecules and maintain membrane fluidity [3]. Due to their ubiquity in nature, the decomposition of biomass and excreta results in an abundance of environmental steroids. This bioavailability of steroids permits their utilization as growth substrates by microorganisms. 2    Figure 1.1. Chemical structures of representative steroids.  Cholesterol is displayed at the bottom with steroidal Rings A to D identified as well as the numbering of carbon atoms employed in this thesis when referring to cholesterol.  1.1.1 Cholesterol The steroid, cholesterol, is characterized by an eight carbon alkyl side chain at C-17, an (S)-hydroxyl at C-3, and a carbon-carbon double bond between C-5 and C-6 (Figure 1.1). First isolated in 1815 by the French chemist M.E. Chevreul from human gall stones, cholesterol consists of up to 45 mol% of total lipids within mammalian cell membranes, making it  the most abundant steroid in mammals. Up to 90% of cellular cholesterol is located within cell 3  membranes in mammals [4].  The C-3 hydroxyl of cholesterol makes the molecule slightly amphipathic permitting polar Van der Waals interactions with the phosphatidyl head groups of membrane lipids, thereby embedding the cholesterol molecule in an ordered orientation within cell membranes. This packing within membrane lipids results in increased membrane fluidity. However, cholesterol distribution within cell membranes is not uniformly distributed, but rather coalesces into regions high in sphingolipids and lipoproteins known as lipid rafts. Consisting of more than 100 lipoproteins, lipid rafts are thought to bring receptors and secondary- messenger molecules into close proximity and have been implicated in T-cell activation [5], cell adhesion [6], and calcium uptake [6]. Depletion of cholesterol from lipid rafts results in the dissociation of lipoproteins and has been shown to disrupt signaling pathways in T lymphocytes [7], enhance Influenza A release from infected cells [8], and decrease fertilization rates in oocytes [9].   De novo steroid biosynthesis (Figure 1.2) is ubiquitous across animals, fungi, and plants and utilizes a similar anabolic pathway [10]. In mammals, for example, cholesterol synthesis starts with the production of isoprenoid units from acetyl-Coenzyme A (AcCoA). These isoprenoid units are converted to isopentyl-5-pyrophosphate and dimethylallylpyrophosphate via the mevalonate pathway, which are condensed to form farnesyl pyrophosphate, geranyl phosphate, and finally, the 30-carbon molecule squalene. Squalene oxygenase incorporates one atom from dioxygen into squalene to produce an epoxide, (3S)-2,3-oxidosqualene. Cyclization of squalene epoxide by oxidosqualene cyclase produces lanosterol which is modified by a series of oxygenases to form cholesterol. In fungi, lanosterol is converted into ergosterol. Phytosterols are synthesized from cycloartenol in plants, which is also generated from squalene epoxide.  Although de novo cholesterol biosynthesis and dietary sources lead to blood cholesterol levels of 5-6 mM in healthy human adults [11], humans, like all animals, are unable to degrade 4  cholesterol and other steroids. Instead, human livers convert up to 1 g of cholesterol to bile acids per day which are then excreted via the small intestine [11].   Figure 1.2. The cholesterol biosynthetic pathway in animals.    5  1.2 Bacterial metabolism of steroids Despite the ubiquity of steroid biosynthesis across animals, plants, and fungi, only a handful of prokaryotes have been demonstrated to undergo de novo steroid synthesis. Prokaryotic steroid biosynthesizers include Gemmata obsuriglobus [12], Methylococcus capsulatus [13], and some myxobacteria [14]. However, advances in metagenomic analyses have led to the identification of oxidosqualene cyclase genes in 34 bacterial genomes from 5 phyla including Bacteroidetes, Cyanobacteria, Planctomycetes, Proteobacteria, and Verrucomicrobia, suggesting that bacterial steroid biosynthesis may be more wide spread than initially thought [15]. Steroid biosynthesis has not been observed in archaea [16].  In contrast to steroid biosynthesis, steroid catabolism is wide spread across at least two bacterial phyla: Actinobacteria and Proteobacteria [17].  Notable steroid-degrading bacteria include Commamonas testosteroni TA441 [18], Nocardia restrictius [19], Steroidobacter denitrificans [20], Pseudomonas putida DOC21 [21], and multiple species of rhodococci [22-24] and mycobacteria [25-27]. Figure 1.3 shows the evolutionary relationship between notable steroid-degrading bacteria as characterized by their 16S rRNA sequences.  Bacterial steroid catabolism was first extensively studied in Nocardia and Mycobacterium sp. by Sih and Lee between 1960 and 1970 [28, 29]. Early observations noted that Nocardia were able to grow on cholesterol as a sole carbon source and, in the process, excrete cholesterol derived metabolites into the culture media [30]. Since the 1970s, studies into bacterial steroid catabolism have focused on  Comamonas testosteroni [18, 31], Rhodococcus jostii RHA1 [22, 25], and Mycobacterium smegmatis MC2155 [26, 32], due, in part, to their potential biotechnological applications of steroid modification [26]. However, in 2007 following the publication of the genome from RHA1, an homologous cholesterol catabolic gene cluster to 6  that of RHA1 was identified in the human pathogen, Mycobacterium tuberculosis (Mtb) [25]. Since 2007, interest into steroid catabolism has emerged due to its correlation with Mtb pathogenesis, as described in Section 1.3.3  [33].     Figure 1.3. Evolutionary relationship between steroid degrading bacteria.   Phylogeny was analyzed from an alignment of 16S rRNA sequences. Yellow, purple, blue, red, and green highlighted regions indicate bacteria belonging to Myobacteria, Rhodococci, Gamma-proteobacteria, Beta-proteobacteria, and Alpha-proteobacteria, respectively. Species are identified as follows: Amycolatopsis mediterranei RB (A_mediterranei), Mycobacterium abscessus ATCC19977 (Mab), Mycobacteria smegmatis mc2155 (M_smegmatis), Mycobacterium tuberculosis H37Rv (Mtb), Mycobacterium intracellulare 1956 (M_intra), Rhodococcus rhodochrous (R_rhodochrous), Rhodococcus jostii RHA1 (RHA1), Rhodococcus equi (R_equi), Thermomonospora curvata DSM 43183 (T_curvata), Actinoplanes missouriensis 431 (A_missouriensis), Sphingomonas wittichii RW (S_wittichii), Novosphingobium sp. PP1Y (N_PP1Y), Novosphingobium aromaticivorans DSM 12444 (N_aromaticivorans), Comamonas tesosteroni TA441 (CNB-2), Burkholderia lata (B_lata), Ralstonia eutropha H16 (R_eutropha),  Cupriavidus necator N-1 (C_necator), Steroidobacter denitrificans DSM18526 (S_denitrificans), Shewanella pealeana ATCC 700345 (S_pealeana), and Pseudomonas putida DOC21 (P_putida). Figure adapted from Bergstand et al. (2016) [17]. 7   Both aerobic and anaerobic steroid catabolism have been described. Anaerobic catabolism is best studied in Steroidobacter denitrificans DSMZ18526 [20, 34, 35]. Anaerobic testosterone catabolism occurs in a primarily β-oxidative manner in which steroid Rings A and B are opened by a series of hydroxylations and retro-Claisen like ring opening reactions [34]. 17-Hydroxy-1-oxo-2,3-seco-androstand-3-oic acid (2,3-SAOA) is diagnostic of the anaerobic pathway as it is not observed in the aerobic pathway [36].  In contrast, aerobic steroid catabolism is well characterized in both proteobacteria and actinobacteria. All aerobic pathways characterized to date are homologous, although specific pathways have divergently evolved to accommodate particular types of steroids. In each case, the catabolism involves up to three distinct processes: β-oxidation of the alkyl side chain, steroid Rings A and B opening by oxygenases, and catabolism of steroid Rings C and D. Each of these processes will be described at length in Sections 1.2.3 – 1.2.5, respectively. Steroid catabolic pathways have been characterized for cholate [18, 22, 31], androgens [18, 37, 38], and cholesterol [25, 39, 40]. Proteobacteria, such as C. testosteroni, are able to grow on testosterone and cholate, although typically not on cholesterol [18]. Actinobacteria, in contrast, often contain multiple steroid catabolic gene clusters. For example, RHA1 contains four such gene clusters [22], encoding for the degradation of cholesterol, cholate, androgens (predicted), and an undetermined steroid. Similarly, R. rhodochrous contains up to five homologs of steroid catabolic genes [41, 42], and M. smegmatis possess at least two [26]. Additionally, gene redundancy in steroid catabolic pathways permits access of steroid-degrading bacteria to modified steroids, as exemplified by Mtb’s ability to oxidize both cholesterol and cholesterol esters [43, 44].   8  1.2.1 Cholesterol catabolic pathway The catabolism of cholesterol is the best described steroidal catabolic pathway in bacteria due in large part, to its involvement in Mtb pathogenesis. Although many of the ~80 genes involved in cholesterol degradation and their operonic structure are conserved across actinobacteria [17], herein, the cholesterol catabolic gene cluster in Mtb will be discussed at length due to: (A) its ubiquity in the literature and (B) the subsequent focus of the research presented herein.  Variations between cholesterol catabolism in other bacteria as well as in the catabolism of different steroids will be addressed in Sections 1.2.7 and 1.2.8.  The Mtb cholesterol catabolic genes are, for the most part, clustered in the genome between rv3494c and rv3574 [25]. Cholesterol catabolic genes have been identified via a combination of transposon mutagenesis looking for insertions in genes that abrogate growth of Mtb on cholesterol [40] and transcriptomics studies looking at the up-regulation of genes during growth on cholesterol [45-48]. Table 1.1 summarizes the data for the cholesterol catabolic gene cluster as well as additional genes involved in cholesterol catabolism. Although the functions of many of these genes remain unknown, parallels between β-oxidative and aromatic degradation pathways have provided the frame work for the catabolic pathway for cholesterol.       9  Table 1.1. Cholesterol catabolic genes in Mtb Locus Synonym Annotation Essential for growth on cholesterol[40] Up regulated on cholesterol[47] Essential for virulenceA rv1106c 3β-HSD 3β hydroxysteroid dehydrogenase rv1130 prpD methylcitrate dehydratase  [49] rv1131 prpC methylcitrate synthase  [49] rv1143 MCR Methyl Acyl-CoA racemase rv3409c choD cholesterol oxidase [50] rv3492c - MCE associated protein rv3493c - MCE associated protein rv3494c mce4F MCE family protein rv3495c lprN MCE family protein rv3496c mce4D MCE family protein rv3497c mce4C MCE family protein rv3498c mce4B MCE family protein rv3499c mce4A MCE family protein rv3500c yrbE4B MCE-associated protein rv3501c yrbE4A MCE-associated protein  [33] rv3502c hsd4A 17-hydroxysteroid dehydrogenase  rv3503c fdxD Ferredoxin rv3504 chsE4 acyl-CoA dehydrogenase rv3505 chsE5 acyl-CoA dehydrogenase rv3506 fadD17 acyl-CoA synthetase rv3515c fadD19 acyl-CoA synthetase  rv3516 echA19 enoyl-CoA hydratase rv3518c cyp142 Cytochrome P450 rv3520c Reductase rv3521 hypothetical protein rv3522 ltp4 ketoacyl-CoA thiolase rv3523 ltp3 ketoacyl-CoA thiolase rv3526 kshA ketosteroid monooxygenase   [51] rv3534c hsaF 4-hydroxy-2-oxovalerate aldolase  rv3535c hsaG acetaldehyde dehyrogenase rv3536c hsaE 2-hydroxypentadienoate hydratase  rv3537 kstD ketosteroid dehydrogenase   [52] rv3538 hsd4B 2-enoyl acyl-CoA hydratase rv3540c ltp2 ketoacyl-CoA thiolase  rv3541c chsH1 Mao-C like enoyl-CoA hydratase    rv3542c chsH2 Mao-C like enoyl-CoA hydratase   [52] rv3543c chsE2 acyl-CoA dehydrogenase  rv3544c chsE1 acyl-CoA dehydrogenase   [52] rv3545c cyp125 Cytochrome P450  10  ARespective gene deletion or transposon mutant in Mtb in referenced study displayed reduced virulence in macrophage and/or animal studies   1.2.2 Cholesterol import Cholesterol is transported across the bacterial cellular envelope by a set of proteins encoded by the mce4 (mammalian cell entry) locus in actinobacteria [54]. In Mtb these genes correspond to the rv3492c to rv3501c locus [33].  The mce4A-mce4F genes are predicted to code for proteins that assemble into a hetero-hexameric ring with a helical, needle-like C-terminal domain that spans the periplasm, connecting the outer and inner membranes [55]. The MCE4 transporter is specific for steroids with long hydrophobic side chains [54, 56]. Although cholesterol import is predicted to be ATP dependent, disruption of the putative ATPase, MceG, or permease subunits of the ATP-binding cassette (ABC) transporter,  YrbE4A, did not Locus Synonym Annotation Essential for growth on cholesterol[40]Up regulated on cholesterol[47] Essential for virulenceA rv3546 fadA5 ketoacyl-CoA thiolase   [47] rv3548c - short chain dehydrogenase  rv3549c - short chain dehydrogenase  rv3550 echA20 enoyl-CoA hydratase rv3551 ipdA CoA transferase subunit A   [52] rv3552 ipdB CoA transferase subunit B  [52] rv3553 ipdC acyl-CoA reductase  rv3556c fadA6 ketoacyl-CoA thiolase  [52] rv3557c kstR2 TetR type repressor rv3559c ipdF ketoacyl-CoA reductase  rv3560c fadE30 acyl-CoA dehydrogenase  rv3561 fadD3 HIP-CoA synthetase  rv3562 fadE31 acyl-CoA dehydrogenase rv3563 fadE32 acyl-CoA dehydrogenase   [52] rv3564 fadE33 acyl-CoA dehydrogenase  rv3565 aspB aminotransferase rv3567c hsaB monooxygenase subunit B rv3568c hsaC DHSA dioxygenase   [39] rv3569c hsaD DSHA hydrolase   [53] rv3570c hsaA 3-HSA monooxygenase   [52] rv3571 kshB Rieske Oxygenase reductase   [51] rv3573c cshE3 acyl-CoA dehydrogenase  rv3574 kstR TetR-type repressor   11  completely prevent growth of Mtb on cholesterol [33]. Similarly, deletion of the mce4 operon in M. smegmatis did not completely prevent cholesterol uptake, suggesting that other mechanisms of cholesterol uptake may exist [56]. In fact, the hydrophobic nature of cholesterol may permit passive diffusion through the inner membrane into the bacterial cell [33].      1.2.3 Side chain degradation  Degradation of the alkyl side chain of cholesterol starts with the oxidation of the terminal carbon at C-26 or C-27 to a carboxylic acid (Figure 1.4). In Mtb, two cytochromes P450 (Cyp) are able to catalyze this step [57, 58]. Cyp125 (Rv3545c) uses dioxygen to oxidize C-26 of cholesterol to generate 5-cholestene-26-oate [58]. The physiological reductase that transfers reducing equivalents from NADH to the Cyp appears to be KshB [58, 59]. Deletion of cyp125 in Mtb CDC1551 leads to the accumulation of cholest-4-ene-3-one upon incubation with cholesterol, suggesting that this metabolite may be the physiological substrate of Cyp125 [60]. Interestingly, in Mtb H37Rv deletion of cyp125 does not disrupt growth on cholesterol due to the presence of a functional Cyp142 which cansubstitutefor Cyp125 in the C-26 hydroxylation of cholesterol [57, 61]. Following formation of 5-cholestene-26-oate, the carboxylate is thioesterified using ATP and CoASH, to 5-cholestene-26-oyl-CoA by the acyl-CoA synthetase, FadD19 permitting the start of β-oxidation of the side chain [62].   12   Figure 1.4 Cholesterol catabolic pathway in Mtb.  The cholesterol catabolic pathway as currently known for Mtb. Enzymes are shown in bold. R1 indicates H or –CH3 for an eight or five carbon side chain, respectively; R2 resprents an unknown side chain or C-17 oxo. Reactions catalyzed by multiple enzymes are displayed as dashed lines. Proposed enzyme reactions are depicted in grey. Metabolite naming for ADD, 3-HSA, DHSA, and DSHA, all represent the C-17 oxo analogs of the depicted structures.    13    β-oxidation of the alkyl side chain involves four repeating steps: desaturation of Cα and Cβ adjacent to a coenzyme A (CoA thioester) by an acyl-CoA dehydrogenase (FadE), hydroxylation of the double bond at Cβ by an enoyl-CoA hydratase (EchA), oxidation of the Cβ hydroxyl to a ketone by a 3-hydroxyl-acyl-CoA dehydrogenase (FadB), and thiolysis of the β-keto-acyl-CoA, releasing an acetyl-CoA or propionyl-CoA group by a β-keto-acyl-CoA thiolase (FadA). In Mtb, cholesterol metabolites with eight carbon CoA side chains, such as 3-oxo-chol-4-en-26-oyl-CoA are preferentially desaturated by the FadE complex of ChsE4-ChsE5 (Rv3504-Rv3505), whereas the five carbon side chain is desaturated by ChsE3 (Rv3573c) [63]. The echA19 gene, located adjacent to fadD19, is up-regulated in the presence of cholesterol and likely encodes the EchA responsible for hydroxylation of eight and five carbon side chains due to the absence of another homolog [25, 47]. Although there are no FadB homologs encoded within the cholesterol catabolic gene cluster, Hsd4A (Rv3502c), annotated as a 17β-hydroxysteroid dehydrogenase, is essential for growth on cholesterol and predicted to dispense for a FadB type enzyme [40, 64]. FadA5 is responsible for the cleavage of the five carbon β-keto-acyl-CoA cholesterol metabolite and may act on the eight carbon side chain as well [65].  Two rounds of β-oxidation, yields the metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4-BNC-CoA) which contains an isopropionyl-CoA side chain at C-17. Genes involved in the removal of the isopropionyl-CoA side chain are clustered together between rv3540c and rv3545c in the igr operon (intracellular growth) which is essential for growth on cholesterol and bacterial persistence in macrophages [66].   β-Oxidation cannot be employed to remove the propionyl-CoA moiety due to the presence of a tertiary carbon at C-17. Removal of the propionyl-CoA moiety involves desaturation by ChsE1-ChsE2 (Rv3544c-Rv3543c) [63] then 14  hydroxylation at C-17 by a unique MaoC-like (R) – enoyl-CoA hydratase, ChsH1-ChsH2 (Rv3541c-Rv3542c) [67]. Retro-aldol C-17/C-22 bond cleavage has been predicted to be catalyzed by an aldolase from the lipid transfer protein (Ltp) superfamily of which  3 are encoded in the Mtb cholesterol catabolic gene cluster: Ltp2, Ltp3, and Ltp4 [25, 68].  Ltp3 and 4 are likely involved in the aldolytic cleavage of C-24 branches in steroids such as β-sitosterol and campesterol, but not cholesterol [68]. Although Ltp2 (Rv3540c) has been implicated in catalyzing this step as ltp2 is adjacent to chsH1, alignments of Ltp2 with other aldolases suggest that Ltp2 may be lacking catalytically relevant residues suggesting that it may display a different role in cholesterol catabolism (Vanderven, 2017; unpublished).  Overall, complete side chain degradation yields two propionyl-CoA and one acetyl-CoA per molecule of cholesterol and generates androstenedione (AD), a metabolite with four steroidal rings and an oxo group at C-17. The previous paradigm stated that side chain degradation completes prior to the start of Rings A and B degradation. Therefore, side chain degradation enzymes were predicted to act on substrates with intact steroidal Rings A and B and conversely, substrates for Rings A and B degradation enzymes were not predicted to possess a CoA thioester. However, a growing body of evidence refutes this paradigm [41, 69], as discussed in Section 1.2.6.   1.2.4 Cholesterol Rings A and B degradation Rings A and B degradation commences with the conversion of cholesterol to cholest-4-ene-3-one [50, 70]. Both 3β-hydroxysteroid dehydrogenase (3β-HSD, Rv1106c) and cholesterol oxidase (ChoD, Rv3409c) have been implicated in catalyzing this reaction based on the function of  homologs [71]. Of the two, only 3β-HSD has been demonstrated to catalyze this reaction in vitro [50, 70]. However, both choD and rv1106c are dispensable for growth of Mtb on 15  cholesterol [72] and deletion of 3β-hsd in Mtb did not reduce growth in guinea pigs [70]. Overall, it is possible that the enzyme responsible for this reaction under physiological conditions has not yet been identified. One possibility is that rv0139, which encodes a putative hydroxysteroid dehydrogenase, may be involved in cholesterol catabolism as it is up regulated in the presence of cholesterol [32]. The catabolism of cholesterol Rings A and B, as well as enzyme and metabolite names are summarized in Figure 1.4. Androstenedione (AD) is converted to androsta-1,4-diene-3,17-dione (ADD) via the 3-ketosteroid Δ1-dehydrogenase, KstD [73, 74]. ADD is then hydroxylated at C-9 by the 3-ketosteroid-9α-hydroxylase, KshAB, resulting in an unstable intermediate which readily aromatizes Ring A and undergoes cleavage of Ring B to form 3-hydroxy-9,10-secondrost-1,3,5(10)-triene-9,17-dione (3-HSA) [29, 59, 75]. 3-HSA is hydroxylated at C-4 by the flavin-dependent monooxygenase, HsaAB, generating a catechol, 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (DHSA) [19, 76]. The extradiol dioxygenase, HsaC, uses dioxygen to catalyze the meta-cleavage of DHSA resulting in a meta-cleavage product (MCP), 4,5-9,10-diseco- α3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid (DSHA) [39]. Finally, the MCP hydrolase, HsaD, cleaves the C-5/C-10 bond of DSHA (using steroid carbon numbering), to generate 2-hydroxy-hexa-2,4-dienoic acid (HHD) and 3aα-H-4α(3′-propanoyl-CoA)-7aβ-methylhexa-hydro-1,5-indanedione (HIP) [77]. HHD is degraded by HsaEFG [25, 78] and HIP feeds into Rings C and D degradation [79]. Rings A and B degradation results in one pyruvate and propionyl-CoA per molecule of cholesterol from the catabolism of HHD. The oxidative manner by which cholesterol Rings A and B are degraded is analogous to the degradation of aromatic compounds such as biphenyl [80]. Interestingly, the identification of the cholesterol catabolic gene cluster in Mtb was due in 16  large part to the observation that Mtb encoded homologs to the extradiol dioxygenase, BphC, and MCP hydrolase, BphD, involved in biphenyl degradation [81, 82]. Homologs of KshA, HsaA, HsaC, and HsaD occur in all known aerobic steroid catabolic pathways [17]. 1.2.5 Cholesterol Rings C and D degradation In contrast to side chain and Ring A and B degradation, the catabolism of cholesterol Rings C and D (HIP) is poorly understood. In the 1960s, Lee and Sih proposed that Rings C and D are degraded either oxidatively, via a Bayer-Villager Monooxygenase (BVMO) and subsequent hydrolysis of the rings, or in a β-oxidative-like manner involving retro- Claisen ring opening reactions [28]. Failure to identify a candidate BVMO in the Mtb genome has implicated the latter pathway proposed by Lee and Sih. Interestingly, Mtb and RHA1 are able to grow on HIP as a sole carbon source suggesting that their respective genomes encode for HIP catabolic genes [79].   The acyl-CoA synthetase, FadD3, was the first characterized HIP catabolic enzyme. FadD3 catalyzes the ATP- and CoASH-dependent thioesterification of HIP to HIP-CoA [79]. Further, deletion of fadD3 in RHA1 caused the extracellular accumulation of HIP in the presence of cholesterol  [79].  Based on the formation of HIP-CoA, Casabon et al. proposed that the catabolism of HIP would be β-oxidative in nature involving CoA thioester metabolites [79]. Although FadD3 is the only HIP catabolic enzyme characterized to date, deletion studies in other bacteria have implicated additional genes. R. equi deficient in fadE30 or ipdAB accumulated extracellular 5OH-HIP or HIP, respectively, upon incubation with cholesterol [23]. Therefore, van der Geize et al. proposed that IpdAB, a predicted CoA transferase, would be involved in HIP-CoA synthesis and FadE30 would desaturate HIP-CoA [23]. Interestingly, fadD3, fadE30, and ipdAB are all part of the KstR2 regulon, a differentially regulated set of genes in the 17  cholesterol catabolic gene cluster [83], as discussed in Section 1.2.7. Lastly, in 1977, Hashimoto and Harakawa observed the accumulation of 5-methyl-4-oxo-octane-1,8-dioate (MOODA) when HIP was incubated with Streptomycetes rubescens [84]. 1.2.6 Concurrent side chain and Rings A and B degradation In 2011, Capyk et al. reported that KshAB, responsible for the aromatization of Ring A, had a higher substrate specificity (kcat/KM) for steroidal substrates that possessed a CoA thioesterified side chain [69]. This suggested that side chain degradation and Rings A/B degradation occur concurrently to some extent. The extent of this concurrent catabolism is unclear. However, it is noted that the substrate specificities of KshAB, HsaAB, and HsaD for substrates with completely degraded side chains are 2-3 log10 orders lower than reported for homologous enzymes and their physiological substrates [59, 76, 77]. The implication is that perhaps these enzymes preferentially act on substrates with partially degraded side chains. Interestingly, FadD3 does not ligate CoASH onto 1β(2′-propanoate)-3aα-H-4aα(3”-propanoate)-7aβ-methylhexahydro-5-indanone (HIP with an isopropionyl side chain at C-17) implying that side chain degradation completes prior to the degradation of cholesterol Rings C and D [79].  1.2.7 Organization and regulation of steroid catabolic gene clusters The cholesterol catabolic gene cluster in Mtb is primarily located between rv3409c and rv3574 [25]. Figure 1.5 depicts the organization of the cholesterol catabolic gene cluster in Mtb. In general, the cholesterol catabolic gene cluster is separated into regions encoding for enzymes that catalyze cholesterol import (rv3492 – rv3501c) or side chain (rv3504 – rv3546), Rings A and B (rv3567c – rv3574), and Rings C and D (rv3548c – rv3564) degradation [25]. Similarly, many of these aforementioned regions are organized into operons which are reflected in their co-regulation on cholesterol [47]. 18  The cholesterol catabolic gene cluster is regulated by at least two TetR family transcriptional repressors, KstR (Rv3574) and KstR2 (Rv3557c) [32, 83], described in Section 1.6. KstR and KstR2 bind DNA at conserved 14 bp palindromic sequences located at the -10 to -35 bp regions of their regulated genes [32]. 19    Figure 1.5. Organization of the cholesterol catabolic gene cluster in Mtb.  Arrows indicate genes involved in cholesterol import (red), side-chain degradation (purple), Rings A and B degradation (green), Rings C and D degradation (blue), unassigned function (black), regulation (yellow), and genes not involved in cholesterol catabolism (grey).  Genes described in length herein are identified in red text.  Figure adapted from  Capyk, J. (2012) [85].20  First characterized by Kendall et al. in 2007 in M. smegmatis [32], the KstR regulon is predicted to comprise at least 74 genes in Mtb based on the occurrence of 16 KstR binding motifs in the genome, of which 13 occur within the cholesterol catabolic gene cluster [32]. Deletion of kstR in M. smegmatis caused the up-regulation of many of the cholesterol catabolic genes however yielded only a slight growth defect [32]. The inducer of KstR was identified to be 3-oxo-cholest-4-en-26-oyl-CoA [86], a cholesterol metabolite generated in side chain degradation (Section 1.2.3). Binding of 3-oxo-cholest-4-en-26-oyl-CoA to KstR results in a conformational change that causes the release of KstR from the DNA binding motif to permit transcription [86]. The first CoA thioester formed in a catabolic pathway is often the effector of that pathway. For example, the effectors of PaaR and FadR from Thermus thermophilus HB8, involved in the catabolism of phenylacetate (PAA) and fatty acids, are PAA-CoA and lauroyl-CoA, respectively [87-89], while the effector of CouR of RHA1, a MarR-type transcriptional repressor,  is p-coumaroyl-CoA [90] The KstR2 regulon is much smaller, comprising only ~14 genes. In Mtb, these genes are clustered between rv3548c and rv3564 in the cholesterol catabolic gene cluster [83]. These genes are predicted to be involved in the catabolism of cholesterol Rings C and D [79]. In 2013, Casabon et al. identified HIP-CoA, the product of the FadD3 reaction, as the effector of KstR2 from Mtb [91]. In RHA1 grown on HIP, genes of the KstR2 regulon were up-regulated, but not those of the KstR regulon [91]. Intriguingly, the KstR2 regulon is highly conserved across steroid degrading actinobacteria and proteobacteria [17, 20, 22]. However, actinobacteria with multiple steroid catabolic clusters only have a single KstR2 regulon, suggesting that the catabolism of all steroids feeds into a shared HIP catabolic pathway [22]. Although genes of the 21  KstR2 regulon are typically conserved between actinobacteria and proteobacteria, they appear to be regulated by a LuxR family transcriptional repressor in proteobacteria [92].  1.2.8 Cholate and testosterone catabolism Cholate and testosterone catabolism is similar to that described for cholesterol, however with a few differences. Testosterone, and more generally androgens, lack a C-17 alkyl side chain and therefore, their catabolic gene clusters only encode for enzymes responsible for the degradation of steroidal rings [18]. Cholate possesses a five carbon side chain at C-17 with a terminal carboxylic acid. Therefore, a cytochrome P450 is not required for activation of the side chain prior to catabolism [18]. As with cholesterol, cholate is thioesterified by an acyl-CoA synthetase, CasG in RHA1, enabling the β-oxidation of the alkyl side chain [62]. Interestingly, catabolism of the cholate alkyl side chain does not involve a FadA5-like β-keto-acyl-CoA thiolase in Pseudomonas putida Chol1, put proceeds through a unique aldehyde intermediate presumably generated by the aldolase, Skt [93].   1.3 Mycobacterium tuberculosis and tuberculosis Mtb, the causative agent of tuberculosis (TB), is responsible for ~1.8 million deaths annually [94]. It is proposed that up to one third of the world’s population are either infected with Mtb and/or will test positive for Mtb exposure, however less than 10% of these individuals will develop TB [94]. Over 60% of the world’s TB cases occur within India, China, South Africa and Russia, with Sub-Saharan Africa having the highest per capita rates of TB [94].  Mtb is an intracellular human pathogen transmitted between hosts via aerosolized droplets resulting from coughing by infected individuals. Once inhaled into the lungs, Mtb is phagocytosed by alveolar macrophages. Using mechanisms yet to be fully understood, Mtb is able to prevent fusion of the phagosome with the lysosome within macrophages, thereby 22  preventing acidification and maintaining an environment permissive for bacterial replication [95]. Mtb is then sequestered by phagocytes into granulomas or tubercles which can occlude the bacteria from the rest of the lung for decades [95]. Active TB results from the breakdown of these granulomas releasing Mtb into the lungs [95]. The exact mechanisms that lead to the development of TB remain unclear, but the immune status of the infected individual plays an important role as the disease is common in the immunocompromised as evidenced by the fact that nearly a quarter of the deaths caused by TB in 2016 were HIV-associated [94]. Streptomycin, the first antibiotic used to treat TB, was first isolated in 1943 by Selman Waksman and his graduate student Albert Schatz [96]. Although initially effective, streptomycin resistant strains of Mtb were observed shortly after its introduction due to its administration as a monotherapy [97]. Thereafter, anti-tuberculosis therapeutics have been prescribed as combination therapies in an attempt to limit the spread of multi drug resistance (MDR). Currently, standard Mtb treatment employs four front line anti-tuberculosis therapeutics, rifampicin (RIF), isoniazid (INH), ethambutol (EMB), and pyrazinamide (PZA), in which all four are administered daily for two months followed by four months of INH and RIF only  [97, 98]. Largely due to the negative side effects of the current drugs and distribution issues within developing nations, MDR-TB has emerged in response to low compliance in completing the standard treatment. Therefore, second line treatments such as the fluoroquinolones and aminoglycosides are increasingly prescribed for the treatment of TB, which has in turn led to widening the spectrum of antibiotic resistance within Mtb [98]. Extensively drug resistant strains, referred to as XDR-TB, are resistant to both isoniazid and rifampicin as well as to at least one of the fluoroquinolones and injectable second line drugs. XDR-TB are estimated to make up almost 10% of MDR-TB cases world-wide [94]. In March 2012, a totally drug resistant Mtb (TDR-TB) 23  was reportedly isolated in India that displayed resistance to all first and second line drugs [99]. Although questions have arisen regarding the validity of this claim by the World Health Organization (WHO), this exemplifies the urgent need for novel therapeutics to obvert further antibiotic resistance in Mtb [97]. 1.3.1 Mycolata Mycobacteria and rhodococci belong to a bacterial taxon known as mycolata. Mycolata are characterized by an outer membrane that contains mycolic acids. Mycolic acids are 2-alkyl, 3-hydroxy long-chain fatty acids consisting of 20 – 100 carbon atoms with various genera and/or species specific desaturations and/or hydroxylations [100]. Mycolata cell walls consist of a thick mycomembrane, composed of a parallel arrangement of mycolic acids linked to arabinogalactan, which is covalently attached to a peptidoglycan layer [100]. The highly hydrophobic nature of the mycomembrane creates a barrier to hydrophilic molecules, giving Mycolata a Gram-negative like characteristic and limiting the effectiveness of many antibiotics [101].  The majority of Mycolata, are non-pathogenic saprophytes often regarded as important organisms due to their vast catabolic capabilities which are employed in bioremediation and biocatalytic applications [80, 102]. For example, the steroid degrading capabilities of Mycolata are being increasingly employed as biocatalysts to produce 17-ketosterols, AD, 9α-OH-AD, and ADD [26, 103-105].  Steroid degradation is ubiquitous across Mycolata [17]. In a recent analysis of 8,200 bacterial genomes, 212 putative steroid-degrading Actinobacteria spp. (including the Mycolata) from 16 genera were identified [17]. Of all mycobacterial and rhodococcal genomes characterized, only M. leprae and Rhodococcus sp. strain AW2509 did not encode a steroid catabolic pathway [17].  24  1.3.1.1 Mycobacterium abscessus  Mycobacterium abscessus (Mab) is a rapidly growing mycobacterium that is involved in nontuberculous pulmonary infections typically within immunocompromised or Cystic Fibrosis (CF) patients [106, 107]. Increased concern has emerged as the global incidence of M. abscessus infections has risen [107]. Due to its intrinsic multi-drug resistance [108], the recent identification of person-to-person transmission of M. abscessus further increases the need for novel therapeutic options [107]. Mab was recently identified as containing a steroid catabolic gene cluster within its genome [17], although the role of steroid catabolism in infection as well as the characterization of said pathway remains unstudied. 1.3.1.2 Mycobacterium smegmatis Mycobacterium smegmatis MC2155 is a largely non-pathogenic mycobacterium that is often used as a model organism in studying Mtb physiology. Compared to Mtb, M. smegmatis grows rapidly and is more genetically tractable permitting wider laboratory use [109]. M. smegmatis grows on cholesterol and contains a similarly organized cholesterol catabolic gene cluster to that of Mtb [17, 26]. A second steroid catabolic gene cluster was recently identified in M. smegmatis that was proposed to be involved in the degradation of androgens as it is not up regulated in the presence of cholesterol, however additional studies are required [27]. 1.3.1.3 Rhodococcus jostii RHA1 Rhodococcus jostii RHA1 is a non-pathogenic, fast-growing bacterium that is genetically tractable. First isolated from lindane-contaminated soils, RHA1 has garnered much interest due to its ability to degrade a wide range of aromatic compounds [110]. The RHA1 genome is over 9.7 Mbp and is one of the largest bacterial genomes sequenced to date [111]. RHA1 contains three complete steroid catabolic pathways, two of which are responsible for the degradation of 25  cholate and cholesterol, respectively [22]. RHA1 has been used as a model organism to investigate cholesterol catabolism [39, 76, 79]. Unlike Mtb, the cholesterol catabolic gene cluster is split into three regions in RHA1 separated by two ~70 kbp segments [111].  1.3.2 Lipid metabolism in Mtb Lipid metabolism in Mtb has emerged as a key area of research due to its crucial role in virulence. The current paradigm states that once inside the phagosome, Mtb is restricted to a limited number of host-derived nutrients of which lipids and cholesterol make up the majority [112]. Therefore, lipids are a primary source of carbon for Mtb during infection. This is reflected in the observation by Cole et al. that the Mtb genome is “overrepresented” in the number of genes predicted to be involved in the catabolism of fatty acids [113]. In fact, disruption of multiple genes involved in lipid metabolism within Mtb are predicted to result in either attenuation or avirulence in a mouse model [114, 115].  The utilization of lipids as a primary nutritional source requires specific metabolic adaptations by Mtb [49]. Catabolism of odd chain fatty acids yield propionyl-CoA  which is toxic at high intracellular concentrations [49, 112, 116]. Propionyl-CoA is metabolized by the methyl citrate cycle (MCC), a central metabolic pathway in Mtb that involves the condensation of propionyl-CoA with oxaloacetate by methylcitrate synthase, PrpC, to make methylcitrate which is fed into TCA by way of isocitrate lyase (Icl) (Figure 1.6) [49]. Disruption of prpC or icl as well as other genes of the MCC prevent growth of Mtb in macrophages [49, 52, 117, 118]. However, a prpDC mutant had no effect on tissue pathology [119]. Similarly, propionyl-CoA is anabolized to virulence-associated polyketide lipids phiocerol-dimycoseroic acid (PDIM), poly-acylated trehaloses (PATS), sulfolipid (SL), and mycolic acids [120]. PDIM, for example, is important in host-pathogen interactions [112]. Lastly, availability of vitamin B12 permits 26  propionyl-CoA metabolism via the methylmalonyl pathway, which can compensate for disruption of the MCC [49, 116].      Figure 1.6. Central metabolic pathways of Mtb involved in fatty acid and cholesterol catabolism.   Diagram displaying how acetyl-CoA, propionyl-CoA, and pyruvate generated from lipid and cholesterol catabolism in Mtb feeds into the methylcitrate cycle (MCC), glyoxylate cycle (GC), and methylmalonyl-CoA pathway. Other acronyms are: FA (fatty acids), MCM (methylmalonyl-CoA mutase), PEP (phosphoenolpyruvate), and 3NP (3-nitropropionate). Figure adapted from Griffin et al. (2012) and used with permission from Elsevier. 27  1.3.3 Role of cholesterol catabolism in Mtb pathogenesis Shortly after the identification of the cholesterol catabolic gene cluster in 2007, it was noted that deletion of many of the genes involved in cholesterol catabolism had been previously identified as essential for growth in macrophages [25, 52]. Mtb strains deficient in cholesterol import due to disruption of genes within the mce4 locus demonstrated severe growth attenuation within mouse lungs [33]. Deletion of the igr locus, involved in the removal of the final propionyl-CoA from the cholesterol side chain, attenuates Mtb growth in macrophages and mouse models [66]. Mtb deficient in hsaC is unable to catabolize cholesterol, displays reduced virulence in mouse models, and causes significantly fewer pulmonary lesions within guinea pigs [39]. This severe attenuation of ΔhsaC Mtb is proposed to result from the accumulation of toxic catechols resulting from the absence of HsaC [39]. Similarly, investigations into Mtb deficient in hsaD, fadA5, kshA, kshB or choD demonstrate growth attenuation in macrophages and/or virulence in mouse models [47, 50, 51, 53]. Mtb deficient in cyp125 display increased sensitivity to azoles, however are not attenuated in macrophages [121]. Furthermore, deletion of ipdAB in the related horse pathogen, R. equi, is patented as a live vaccine [23]. Overall, deletion of 17 of the ~80 genes involved in cholesterol catabolism have demonstrated some attenuation in infection models, strongly implicating a role of cholesterol catabolism in Mtb virulence (Table 1.1). Unexpectedly, many of the aforementioned deletion strains in Mtb are unable to grow in the presence of cholesterol and a second carbon source suggesting the accumulation of toxic cholesterol metabolites. Cholesterol is readily available to Mtb during infection. Granulomas are rich in cholesterol and cholesterol esters [122]. During infection, Mtb extracts host-derived cholesterol from the phagosomal membrane [123]. Interestingly, individuals that display lower blood 28  cholesterol levels are less likely to develop TB [124, 125]. Additionally, patients receiving statins to lower blood cholesterol levels display a lower risk for TB [126, 127].  Although Mtb grows on cholesterol as a sole carbon source in vitro and disruption of cholesterol catabolism impairs pathogenicity, the exact function of cholesterol catabolism in virulence is poorly understood. Unexpectedly, growth of Mtb on 14[C]-4 or 14[C]-26 – cholesterol yielded radioactivity in primarily CO2 and membrane lipids, respectively [33, 49]. This suggests that production of virulence lipids, such as PDIM, may depend on the cholesterol side chain. In addition to its use as an intracellular carbon source, there are two other primary hypotheses concerning the role of cholesterol catabolism in Mtb. Firstly, it is possible that cholesterol is required for Mtb uptake by macrophages. Chemical depletion of cholesterol from lipid rafts in macrophages prevent Mtb uptake [128]. Additionally, cholesterol in the phagosomal membrane is essential in preventing the phagosome-lysosome fusion in mice [129]. Secondly, cholesterol catabolites may play a role in modulating host-pathogen interactions. For example, catabolism of the alkyl side chain of cholesterol by Mtb involves the formation of various hydroxylated cholesterol intermediates, some of which are structural analogs to immune-regulating steroids [59, 130]. Furthermore, degradation of cholesterol Rings A and B by Mtb generates structural analogs of cholecalciferol, or Vitamin-D3. Vitamin-D3 has broad immunological regulatory roles during Mtb infection and modulates host lipid metabolism [131]. Although it is unknown whether Mtb is able to excrete these catabolites to modulate host response, precedence exists for such activity within mycobacteria. Mycobacterium leprae is an intracellular pathogen that is unable to utilize cholesterol as a nutrient source [132]. However, M. leprae converts host derived cholesterol to cholestenone during infection, which is excreted into the macrophage [132]. Marques et al. propose that 29  cholestenone, which is the preferred substrate for human Cyp27A1 [133], is readily converted to 27-hydroxycholestenone in the macrophages, which modulates immune response [132].      1.3.3.1 Cholesterol catabolism as a target for novel therapeutics Due to the central role of cholesterol catabolism in the virulence of Mtb, and the potential for toxic cholesterol metabolites, numerous studies have attempted to identify small molecules that inhibit the cholesterol catabolic pathway directly [53, 134-136]. In their preeminent study, VanderVen et al. screened a chemical library for compounds that specifically inhibit Mtb growth in macrophages identifying a disproportionally high number of hits that target cholesterol catabolism [134]. A total of 41 compounds inhibited intracellular growth of Mtb and prevented in vitro growth of Mtb only in media containing cholesterol [134]. Of the characterized compounds, one targeted PrpC, involved in the MCC, one acted on the adenylate cyclase, Rv1625c, whose role in cholesterol catabolism is unclear, and two inhibited the HsaAB reaction with IC50 values of <10 μM [134]. HsaD has also seen much attention as a target for anti-tuberculosis compounds due to its essentiality in virulence, absence of homologs in humans, and ease of  use in high throughput screening [53, 77, 135]. Recently, 7 compounds were identified that both inhibited HsaD in vitro and prevent growth of Mtb in media containing cholesterol [53]. These inhibitors were shown to bind within the HsaD active site in x-ray crystal structures [53]. Although reported IC50 values were relatively high (>100 μM), this study indicates that screening for inhibitors of specific cholesterol catabolic enzymes can be effective in identifying compounds with anti-tuberculosis activity in vivo [53]. 30  1.4 Coenzyme A (CoA)  Figure 1.7 Structure of Coenzyme-A. Specific moieties are identified on the bottom and right side. Blue and red dotted lines indicate fragments that generate the indicated molecular weights upon in source fragmentation during LC-MS/MS.  During the initial characterization of the cholesterol catabolic pathway, the role of coenzyme A (CoA) thioesters was thought to be limited to the catabolism of the alkyl side chain via β-oxidation. However subsequent studies involving KshAB, FadD3, KstR, and KstR2 have suggested that CoA thioesterified steroidal substrates may play a larger role in cholesterol catabolism than initially thought [69, 79, 86]. The body of work presented herein provides a much expanded role for CoA thioesters in cholesterol catabolism. In brief, CoA thioesters are shown to (A) determine substrate specificities of cholesterol catabolic enzymes, (B) regulate cholesterol catabolism, and (C) make up the majority of metabolites in the catabolism of Rings C and D. CoA, or CoASH in its non-thioesterified form, is an essential cofactor involved in a number of cellular processes in all known organisms [137]. It is estimated that up to 4% of all 31  known enzymes require CoA as a cofactor [137]. CoA consists of three moieties: β-mercaptoethylamine, pantothenate, and adenosine diphosphate (ADP) (Figure 1.7). CoA is synthesized in five steps in Mtb: 1) phosphorylation of pantothenate (Vitamin B5) to 4’-phosophopantothenate by pantothenate kinase (PanK or CoaA); 2) condensation with a cysteine residue by phosphopantothenoylcysteine synthetase (CoaB); 3) decarboxylation by phosphopantethenoylcysteine decarboxylase (CoaC); 4) adenylation by phosphopantetheine adenylyltransferase (CoaD) to form dephosopho-CoA; and 5) phosphorylation by dephosphocoenzyme A kinase (CoaE) to CoA [138].  CoaB and CoaC are fused as a bifunctional enzyme in Mtb [139]. CoaA, CoaBC, and CoaE are essential for Mtb survival [140, 141].  CoA has two primary functions: acting as an acyl group carrier and activating carbonyl groups. CoA thioesterification sequesters metabolites within the cell due to their inability to cross the hydrophobic cell membranes. Similarly, CoA increases the solubility of hydrophobic molecules. The CoA moiety is used to target specific acyl groups to their cognate enzymes in numerous anabolic and catabolic processes  [142]. CoA functionality is dependent upon its free thiol group. Deprotonation of the thiol generates a strong nucleophile that is able to attack the carbonyl carbon of carboxylates or ketones resulting in a CoA thioester. Due to their relatively high standard free energies of hydrolysis, CoA thioesters have a high acyl group transfer potential [143]. Thus, the acyl group attached to CoA is described as ‘activated’. For example, the synthesis of citrate from acetyl-CoA and oxaloacetate by citrate synthase is thermodynamically driven by the hydrolysis of acetyl-CoA [142]. Similarly, the formation of a CoA thioester results in increased acidity of adjacent protons, as compared to esters or carboxylic acids, permitting aldol or Claisen condensation reactions [144]. Finally, some enzymes are known to utilize the binding energy from noncovalent interactions with CoA to 32  decrease activation energy of energetically unfavourable reactions, thereby increasing the rate of catalysis [145-147].    Due to their ubiquity in metabolic processes, methods to identify and quantitate CoA thioesters have become paramount in lipidomic and metabolic research. High performance liquid chromatography (HPLC) of CoA thioesters has been broadly employed due to their characteristic absorbance at 258 nm (ε258 = 11.9 mM-1 cm-1) from the adenine moiety [79, 148]. Chromatography is obtained in reverse phase typically with C18 or pentafluoridated phenyl (PFP) resins using an ammonium acetate or ammonium formate counter ion on the mobile phase due to the hydrophilicity of the CoA moiety [69, 79, 149, 150]. Mass spectrometry (MS) of CoA thioesters is typically performed using electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) due to low volatility and instability of the CoA moiety [149]. Recently, multiple reaction monitoring (MRM) has become prevalent in LC-MS analysis of CoA thioesters due to their characteristic fragmentation [149, 151]. Voltage induced in-source fragmentation of CoA thioesters results in a [428]+ m/z fragment as well as a loss of 507 fragment [M+H-507]+ m/z fragment (Figure 1.7) [151]. Therefore, highly sensitive and precise targeted analysis of CoA thioesters is obtained by monitoring the [M+H]+ → [428]+ and [M+H]+ → [M+H-507]+  transitions. Using MRM, CoA thioesters have been reportedly detected in the sub nanomolar range from biological samples, although sensitivity is highly sample and instrument dependent [149, 151]. 1.5 Meta-cleavage product (MCP) hydrolases HsaD, the cholesterol catabolic enzyme responsible for the hydrolysis of DSHA to HIP and HHD, is a meta-cleavage product (MCP) hydrolase. MCP hydrolases hydrolyze carbon-carbon bonds in vinylogous-1,5-diketones [152] and, with the exception of LigY [153], belong to 33  the α/β hydrolase superfamily. Genes encoding MCP hydrolases are prevalent in aromatic degrading bacteria, and tend to be organized into an operon with extradiol dioxygenase genes [154, 155]. MCP hydrolases have been separated into groups on the basis of their substrate specificities. Type I, II, and III MCP hydrolases act on the cleavage products of bicyclic, monocyclic, and heteroaromatics, respectively. To date, the best characterized MCP hydrolases are BphD from Burkholderia xenovorans LB400 and MhpC from Escherichia coli, which  transform 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) and 2-hydroxy-6-oxo-nona-2,4-diene- 1,9-dioate, respectively [81, 156-159].  1.5.1 Structure of MCP hydrolases MCP hydrolases contain a conserved Ser-His-Asp catalytic triad [160] located adjacent to an oxyanion hole, characteristic of other hydrolytic enzymes [156, 161]. The α/β hydrolase fold consists of seven parallel and one antiparallel β-strand, twisted into a β-sheet surrounded by six alpha helices. An α-helical ‘lid’ domain is inserted between β6 and α6 of the core domain and contributes to substrate specificity [162]. The active site is an elongated tunnel located within the core domain and underneath the lid domain containing two subsites: a polar one binds the dienoate moiety of the MCP and a hydrophobic one binds the remainder of the MCP [77, 81, 162]. In HsaD, the entrance to the active site pocket is enlarged compared to BphDLB400 and MhpC, with a single polar region located within 10 Å of the active site whose function is currently unknown [77].  1.5.2 Mechanism of serine-dependent MCP hydrolases Early data had been interpreted to favour an acid/base mechanism of C-C bond cleavage in Ser-dependent MCP hydrolases [157]. However, recent evidence indicates that these enzymes employ covalent catalysis [156, 159]. Thus, when BphDLB400 was rapidly quenched in the 34  presence of HOPDA, a benzoyl-Ser112 acyl enzyme intermediate was observed by ESI-MS [158]. The acylated species was also observed in the H265Q variant of BphD in cristallo [158]. Deacylation of the benzyl-Ser112 intermediate incorporated a single 18O equivalent (from H218O) into the product, benzoate, and proceeded at a rate consistent with HOPDA turnover [158]. This observation contrasted work done on MhpC in which two 18O were observed in reaction products, supporting the reversible formation of a gem-diol intermediate [157]. Interestingly, the catalytic His265 in BphDLB400 is not required for acylation of Ser112 but is essential for deacylation, further supporting covalent catalysis [158]. Consequently, deprotonation of the catalytic serine (Ser112 in BphDLB400 or Ser114 in HsaDMtb) appears to be substrate-assisted and facilitated by localization of an electron lone pair adjacent to the C-6 carbonyl (Figure 1.8) [159]. In summary (Figure 1.8), the alkoxide-like catalytic serine, generated by substrate-assisted deprotonation, acts as a nucleophile to attack the C-6 carbonyl and generated a tetrahedral intermediate. This intermediate collapses, causing C-C bond cleavage, release of HHD and an acyl-enzyme intermediate. The latter undergoes deacylation via hydrolysis to release a carboxylate: HIP and benzoate in the case of HsaD and BphDLB400, respectively.   Mechanistic studies of MCP hydrolases have been facilitated by the characteristic yellow colouration (λmax, HOPDA = 405 nm; λmax, DSHA = 392 nm) of the MCP and the use of spectrophotometric techniques [39, 77, 159]. Interestingly, mutation of the catalytic serine to alanine in MCP hydrolases results in a orange-coloured intermediate (ESred) which, in the HsaD and DSHA system, absorbs at 459 nm [77]. Ruzzini et al. proposed that ESred develops from torsion of the MCP π orbital system coupled with the lone pair localization at C-5. However, the exact nature of ESred is unknown [159]. Titration of these catalytically inactive MCP hydrolases 35  into MCPs permits the measurement of dissociation constants (KD) by following the increase in ESred formation [77].  Figure 1.8 Mechanism of HsaD and MCP hydrolases.  R represents the Rings C and D moiety in DSHA for the HsaD catalyzed reaction or could represent a phenyl group in reactions catalyzed by BphD or DxnB2.   1.6 TetR family of transcriptional repressors The cholesterol catabolic gene cluster is regulated by two TetR family repressors (TFR): KstR and KstR2 [83]. TFRs are named after a founding member involved in regulating tetracycline resistance [163] and is one of the most widely distributed families of transcriptional regulators in bacteria [164-166]. Despite significant sequence variation, the proteins form a conserved L-shaped α-helical structure featuring an N-terminal DNA-binding domain (DBD) and a larger C-terminal effector-binding domain (EBD) [164]. The DBD contains a helix-turn-helix motif involved in binding to operator DNA. TetR protomers associate into homodimers or higher oligomers that recognize palindromic sequences in the operator DNA. More specifically, an α-helix of the TetR DBD called the recognition helix forms specific electrostatic and aromatic contacts in the major groove of the DNA of the operator sequence [164]. The vast majority of 36  TFRs are repressors in the absence of their effector. Binding of the effector triggers a conformational change that shifts the position of the recognition helix, resulting in release of operator DNA by the regulator. Despite a generally conserved structure and mechanism of action, the specific position of the ligand-binding pocket in TFRs and its chemical composition vary dramatically, resulting in specific responses to a vast assortment of small molecules. This variation also makes it difficult to predict the chemical nature of the cognate ligand, necessitating the characterization of individual family members. 1.7 Coenzyme A transferases 1.7.1 The ipdAB genes encode a predicted CoA transferase IpdAB, a predicted Class I CoA transferase (CoT), has recently garnered much attention due to its involvement in Mtb pathogenesis. Based on transposon mapping studies, ipdA and ipdB were predicted to be essential for the growth of Mtb in macrophages [52]. Interestingly, an ipdAB mutant in the related horse pathogen, Rhodococcus equi has been patented as a live vaccine for use in foals [23]. The ipdAB genes are also predicted to be essential for in vitro growth of Mtb on cholesterol based on Tn-seq studies [40].  The ipdAB genes are located within the KstR2 regulon and are strongly implicated in the catabolism of HIP [23, 91]. Both genes are up-regulated in the presence of HIP [91] and incubation of R. equi deficient in ipdAB accumulated 5OH-HIP in the supernatant causing the authors to predict that IpdAB would be involved in the production of 5OH-HIP-CoA via transferring a CoA moiety from a CoA donor such as acetyl-CoA or succinyl-CoA [23]. However, with the discovery that CoA thioesterification of HIP is catalyzed by FadD3 [79], it is unclear what role a CoA transferase would have in the catabolism of HIP. 37  1.7.2 Classes of CoA transferases CoTs catalyze the reversible transfer of CoA from a thioester to a free carboxylate [143, 167]. Three classes of CoTs have been identified, the first two of which belong to the same superfamily. In Class I CoTs, CoA is transferred from the acyl group of the donor substrate to a free carboxylate of an acceptor substrate, typically a small organic acid (see Section 1.7.3) [168-170]. Class II CoTs are subunits of the citrate and citramalate lyase complexes, and catalyze the hydrolysis of small CoA thioesters using citrate or citramalate in a partial mechanism analogous Class I CoTs [171, 172]. Although Class III CoTs belong to a different superfamily, they appear to utilize a mechanism similar to that of Class II CoTs [173, 174].  Class I CoT make up the most diverse class of CoT as they are found in a wide range of catabolic and anabolic pathways [167]. These enzymes are widely promiscuous, displaying CoA transferase activity between an array of CoA donors, such as acetyl-CoA, propionyl-CoA or succinyl-CoA, and small acids, such as acetate, propionate, or succinate [143, 169, 175]. Although countless studies have involved expressing and purifying Class I CoT from across all kingdoms of life, three have been characterized in detail and are representative of the family: Succinyl-CoA: 3-oxoacid Coenzyme A transferase (SCOT) from pig heart is involved in the metabolism of ketone bodies and forms acetoacetyl-CoA from succinyl-CoA [143]; the β-ketoadipate:succinyl-CoA transferase from Pseudomonas putida (PcaIJ), involved in aromatic degradation; and the glutaconate CoA transferase from Acidaminococcus fermentans (GCT).  1.7.3 Structure of Class I Coenzyme A transferases Class I CoTs are formed from two subunits typically assembled as an α2β2 heterotetramer [169]. However in some CoT, such as SCOT, the α and β subunits are fused into a single enzyme which displays an α4 oligomeric state [147]. The α and β subunits appear to evolutionarily related 38  as they display similar, but not identical, folds, however they often share low amino acid identity. The α subunit consists of a seven stranded parallel β-sheet sandwiched between seven α-helices, whereas the β subunit contains a six stranded (5 parallel, one antiparallel) β-sheet sandwiched between six α-helices [147, 169]. The active site of CoT occurs between the α/β dimeric interface and is formed from catalytic residues from both subunits. The active site pocket is typically connected to a long channel following the contours of the dimeric interface to an adenine binding pocket located near the enzyme’s surface [147]. X-ray crystal structures of substrate bound Class I CoT show the acyl moiety bound within the active site with a highly conserved catalytic glutamic reside located less than 3 Å from the thioester carbonyl and the CoA moiety binding along the channel [147]. 1.7.4 Mechanism of Class I Coenzyme A transferases Class I CoT utilize a well characterized and highly conserved ping-pong mechanism in the transfer of a CoA moiety from a CoA donor to a small acid involving a highly conserved catalytic glutamic acid residue summarized in Figure 1.9 [143, 169, 170]. In this reaction, the conserved glutamic acid acts as a nucleophile, subsequently forming a glutamyl-CoA-enzyme intermediate prior to CoA transfer to a small acid [143, 175]. Formation of the glutamyl-CoA intermediate causes the release of the acyl group from the CoA donor, prior to binding of the acceptor acid within the active site which deacylates the catalytic glutamate [175]. In all Class I CoT characterized to date, the conserved glutamate is located in the β subunit or domain of the α2β2 and α4 oligomers, respectively, and occurs at the back of the active site [167, 169, 175, 176]. The glutamyl-CoA intermediate has been trapped using sodium borohydride (NaBH4) which reduces the thioester carbonyl of the glutamyl-CoA intermediate to a hemithioacetal [143]. Therefore, Class I CoA transferases are irreversibly inhibited by NaBH4. 39   Figure 1.9 Mechanism of CoA transferases. Red coloured species represents the glutamyl-CoA intermediate.   1.8 Aim of this study The aim of this study is to elucidate metabolic and regulatory steps involved in the catabolism of cholesterol Rings C and D by Mtb and other actinomycetes. Chapters 2 – 5 describe four different studies related to this aim, and which together develop the central theme that CoA thioesters are essential in the catabolism of the last half of the steroid molecule.  Chapter 2 describes the role of CoA thioesters in determining the substrate specificity of the MCP hydrolase, HsaD. This work extends the observation by Capyk et al. (2011) that cholesterol side chain and Rings A and B degradation can occur concurrently to some extent in Mtb [69]. HsaD from Mtb and Mab_3810, the putative HsaD homolog, from M. abscessus were expressed heterologously and purified from E. coli. CoA thioesterified substrates were generated enzymatically from metabolites accumulating in a hsaC deficient strain of RHA1. Experiments relating to growth and gene expression of Mab were performed by Kirstin Brown. Overall, data for the completion of Rings A and B degradation prior to side chain degradation in at least some 40  mycobacteria is presented via the characterization of the recently identified steroid catabolic gene cluster in the related pathogen M. abscessus.  Chapter 3 describes the mechanism by which the cholesterol catabolite, HIP-CoA acts as an effector of KstR2. This work builds on the identification of HIP-CoA as the effector of KstR2 by Casabon et al. (2013) and is adapted from Crowe, et al. (2015) [91, 177]. More specifically, the strength of KstR2:HIP-CoA complex was determined using isothermal titration calorimetry (ITC) and a mechanism for HIP-CoA mediated regulation of KstR2 was proposed via the comparison of X-ray crystal structures of substrate-free KstR2 from RHA1 (KstR2RHA1)  and KstR2Mtb·HIP-CoA. Experiments relating to the crystallization and structure determination of KstR2:HIP-CoA were performed by Peter Stogios. Chapter 4 describes the elucidation of HIP catabolism using molecular genetics, metabolomics and biochemical approaches. KstR2 regulon genes were deleted in Mtb, RHA1, and/or M. smegmatis. Metabolites that accumulated in the presence of cholesterol were characterized using a combination of mass spectrometry, chemical synthesis, and nuclear magnetic resonance (NMR). This involved developing a method to purify and enrich CoA thioesters. Five KstR2 regulon enzymes were expressed heterologously, purified to homogeneity, and used to reconstitute the HIP catabolic pathway in vitro. Overall, the enzymes responsible for opening of cholesterol Rings C and D were identified and a near complete pathway for HIP catabolism is proposed. Much of the LC-MS experiments were performed by Dr. Israel Casabon and were aided by Jason Rogalski and Dr. Leonard Foster at The University of British Columbia. Experiments involving the culturing of Mtb were performed by Kirstin Brown and Jie Liu. The contents of this chapter were published in mBio in 2017 [178]. 41  Chapter 5 provides the first biochemical and structural characterization of IpdAB. IpdAB, previously annotated as a CoA transferase, was identified as the enzyme responsible for opening cholesterol Ring C from the study described in Chapter 4 [178]. Further analysis of the enzyme demonstrated that IpdAB does not possess CoA transferase activity, but rather catalyzes the hydrolysis of cholesterol Ring C via a retro-Claisen like mechanism. A combination of X-ray crystal structures of substrate-free and substrate-bound IpdAB from RHA1 (IpdABRHA1), oligonucleotide-directed mutagenesis, stable isotopic labelling, and NMR was employed to characterize the mechanism by which IpdAB opens Ring C. The crystallization of IpdAB and structure determination was performed by Sean Workman, Liam Worrall, and Nobuhiko Watanabe from the Strynadka Lab at UBC.  Overall, this thesis describes a series of steps in the catabolism of cholesterol by Mtb beginning with the hydrolysis of the Ring A-opened MCP, DSHA, to the Rings C/D-opened metabolite MOODA-CoA. Similarly, the mechanism of regulation for these steps is described. The work included in this thesis contributes to the understanding of bacterial steroid degradation and provides a frame work for developing novel anti-tuberculosis therapeutics targeting cholesterol catabolism.  42   Steroid catabolism in M. abscessus provides insights into the order Chapter 2:of cholesterol Rings A and B and sidechain degradation in actinobacteria 2.1 Introduction The classical paradigm of cholesterol catabolism states that the alkyl side chain is degraded prior to Rings A and B. However, work by Capyk et al. (2011) demonstrated that the Rings A and B degrading enzyme, KshAB, preferentially act on CoA thioesterified substrates, thereby indicating that side chain and Rings A and B degradation occur concurrently to some extent [69]. In contrast, Casabon et al. (2013) showed that side chain degradation must be completed before Rings C and D degradation occurs [79]. These studies established that the intervening enzymes, HsaAB, HsaC, and HsaD, could act on substrates with incompletely degraded side chains. However, the extent to which side chain and Rings A and B degradation occurs concurrently is unclear. Herein, the order of cholesterol side chain and Rings A and B degradation was investigated in Mtb, the related pathogen, M. abscessus (Mab), and RHA1. The ability of Mab to grow on different steroids was determined. Comparative bioinformatic analyses of the steroid catabolic clusters in Mab, Mtb and M. smegmatis identified differences in orthologs of HsaD, the last enzyme involved in Rings A and B degradation, suggesting that the order of cholesterol side chain and Rings A and B degradation differs between mycobacterial species. Similarly, RHA1 deficient in hsaC and hsaD accumulated Rings A/B catabolites with partially degraded side chains. The substrate specificity of HsaD from Mtb (HsaDMtb) and Mab (HsaDMab), were compared for steroids with and without a completely degraded side chain. The results from steady-state kinetic analyses were validated by determining the dissociation constant (KD) of a 43  catalytically inactive variant of HsaDMtb. Overall, we present the first in vitro evidence for steroid utilization by Mab and provide a new order of side chain and Rings A and B degradation of cholesterol in mycobacteria.   Experiments relating to the growth of Mab were performed by Kirstin Brown. All other experiments were performed by myself. Some of this chapter was prepared as a manuscript for submission in 2018 as: Crowe, A. M, Brown, K., Kulkarani, J., Yam, K., and Eltis, L. D. (2018) The unusual convergence of steroid catabolic pathways in Mycobacterium abscessus.  2.2 Materials and methods 2.2.1 Chemicals and reagents CoASH and ATP were purchased from Sigma-Aldrich (St. Louis, MO). All reagents used were of HPLC or analytical grade. Phusion High-Fidelity DNA polymerase, restriction enzymes, and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA). CasI, an acyl-CoA ligase, was purified as previously described [179]. Oligonucleotides were purchased from Integrated DNA Technologies (San Diego, CA). Water for buffers was purified using a Barnstead Nanopure DiamondTM system (Dubuque, IA) to a resistivity of at least 18 megaohms. 2.2.2 DNA manipulation DNA was propagated, purified, amplified and cloned using standard techniques [180]. Expression plasmids for HsaDMtb and its S114A variant were described previously [77]. The hsaDMab gene (mab_3810) was amplified from M. abscessus ATCC 19977 genomic DNA using primer sequences 5’-GAACCATATGGACGTGACATACGAGGGCACCA-3’ and 5’-CCCAAGCTTTTAGCGCCGCAAAAAGTCGACG-3’. The amplicon was digested with NdeI and HindIII and ligated into pET41b(+) (EMD Millipore) generating pETHDMAB. DNA was 44  sequenced by Genewiz (South Plainfield, NJ). Plasmids were transformed into Escherichia coli Rosetta 2 (DE3) (EMD Millipore) by electroporation using a MicroPulser from Bio-Rad (Hercules, CA). The hsaC and hsaD (RHA1_RS22130) deficient strains of RHA1 were generated as previously described [25]. 2.2.3 Phylogenetic analyses Phylogenetic analyses were performed using the PhyML server available on the phylogeny.fr web service [181, 182] using structure-based alignments of meta-cleavage product (MCP) hydrolases generated by TCOFFEE ESPRESSO [183, 184]. Phylogenetic tree figures were prepared using FigTree v1.4.3.  2.2.4 Growth of bacterial strains Mab ATCC19977 was grown on M9 minimal media + 0.5% tyloxapol supplemented with either 0.5 mM cholate, 0.5 mM 4-AD, 0.5 mM cholesterol, or 4.5 mM glycerol at 37oC. Growth was measured by optical density at 600 nm and by CFU enumeration. RHA1 WT and mutants were grown on solid media containing M9G and biphenyl. Single colonies were used to inoculate 50 ml of M9G [185] containing 20 mM sodium pyruvate, and cultures were grown at 30oC to turbidity (~3 d). For protein production, E. coli Rosetta 2(DE3) containing pT7HD1, pEMHSA, or pETHDMAB were grown in 1 L LB supplemented with 100 μg ml-1 ampicillin (pT7HD1 and pEMHSA) or 25 μg ml-1 kanamycin (pETHDMAB) and 34 μg ml-1 chloramphenicol at 37oC and 200 rpm. At mid log (OD600 = 0.6), isopropyl β-D-1-thiogalactopyranoside was added to 0.5 mM to induce hsaD expression. Cells were immediately transferred to 30oC, grown overnight, then harvested by centrifugation at 4oC (4,000 g, 15 min) and frozen at -80oC until use. 45  2.2.5 Metabolite analysis Metabolites were analyzed in the culture supernatants of pyruvate-grown RHA1 cells incubated with cholesterol and of steroid-grown Mab. For RHA1, cells were grown to mid log (OD600 = 0.6) on 500 ml M9G containing pyruvate, harvested by centrifugation (4000 × g, 16oC, 25 min), suspended in 50 ml M9G containing 0.5 mM solid cholesterol and incubated at 30oC for 24 h in 250 ml baffled flasks. At regular time intervals, aliquots of 1 ml were withdrawn and cells were removed by centrifugation. To extract metabolites, 750 μl of the supernatant was acidified using 25 µl glacial acetic acid and was mixed vigorously with 750 μl of ethyl acetate. 5α-Cholestane was added to 26 µM as an internal standard. Supernatants of Mab cultures were handled similarly except that 0.3 ml aliquots were taken, 0.16 mM 5α-cholestane was added, samples were acidified with 10 µl glacial acetic acid, and metabolites were extracted with 0.3 ml ethyl acetate. Organic layers were dried under nitrogen, suspended in 50 μl pyridine and derivatized with trimethylsilane (TMS). Samples were analyzed by gas chromatography coupled-mass spectrometry (GC-MS) as previously described [76].  2.2.6 Purification of HsaCMtb, HsaDMtb, HsaDMtb S114A, and HsaDMab  HsaCMtb was purified as previously described [39]. HsaDMtb, HsaDMtb S114A, and HsaDMab were purified as previously described for HsaDMtb using the following modifications [77]. Following purification using Source15 Q anionic exchange resin (GE Healthcare), HsaD-containing fractions were concentrated to ~4 mg ml-1. HsaDMtb (WT and S114A) were brought to 1 M ammonium sulphate in 20 mM HEPES, pH 7.5 then loaded onto 8 ml of Source15 Phenyl resin (GE Healthcare) and eluted over a gradient of 1 to 0 M ammonium sulphate in 20 mM HEPES, pH 7.5 over 20 column volumes. HsaD-containing fractions were pooled, concentrated to ~10 mg ml-1 using a stirred cell concentrator equipped with a 10K amicon ultracentrifugation 46  membrane (EMD Millipore) then flash frozen in liquid nitrogen and stored at -80oC until use. HsaDMab was brought to 1.3 M ammonium sulphate in 20 mM HEPES, pH 7.5 causing a white precipitate to form. After incubating for 1 h at 4oC, the HsaDMab precipitate was harvested by centrifugation (4,000 g; 10 min) and the pellet was solubilized in 20 mM HEPES, pH 7.5. HsaDMab was exchanged into 20 mM HEPES, pH 7.5, then concentrated to 16 mg ml-1 using a 10K Amicon UltracelR unit (Merck-Millipore). All proteins were prepared to greater than 95% apparent homogeneity as assessed by SDS PAGE analysis. Protein concentrations were determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) using the manufacturer’s protocols. 2.2.7 Preparation of substrates 3,4-Dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (DHSA) and 3,4-dihydroxy- 17-isopropinoyl- 9,10-seconandrost-1,3,5(10)-triene-9-one (DHSBNC) were prepared from cholesterol using an hsaC mutant of RHA1 as a biocatalyst as described previously with the following revisions [39]. Supernatant extracts containing DHSA and DHSBNC were loaded onto a Hewlett Packard Series 1100 HPLC system equipped with a Luna 5u Silica (2) 100A HPLC column (Phenomenex) operated at 1 ml min-1. DHSA and DHSBNC were separated using a 28 ml gradient of 100% hexane and 0.5% acetic acid into 28% ethyl acetate and 0.5 % acetic acid. DHSBNC and DHSA eluted at 22 and 26 min, respectively. The purity and identity of DHSA and DHSBNC were verified using GC-MS on an Agilent 6890 series GC equipped with an HP-5ms 30 m x 250 μm capillary column (Hewlett Packard) as described previously [39].  DHSBNC-CoA was produced by reacting 1 mM DHSBNC with 1.25 mM CoASH, 1.5 mM ATP, 5 mM MgCl2, and 2 μM CasI in 20 mM HEPES, pH 7.5 at 22oC for 1 hour. The 47  resulting DHSBNC-CoA was purified by HPLC using a Luna 3u PFP(2) 100A (50 x 4.6 mm) column in a gradient of 100 mM ammonium acetate, pH 4.5 into 100 mM ammonium acetate, pH 4.5, 90% methanol, over 20 min. DHSBNC-CoA eluted at 18 min. Methanol was removed from the purified DHSBNC-CoA under nitrogen and the sample was diluted 1:3 with water then desalted using a Strata-X 33u 30 mg solid phase extraction column (Phenomenex) and eluted in methanol using the manufacturer’s protocols. The mole recovery of DHSBNC-CoA was ~80%.  DSHA, 4,5-9,10-diseco-3-hydroxy-5,9-dioxo-23,24-bisnorchola-1(10),2-dien-4,22-dioate (DSHBNC), and DSHBNC-CoA were generated by incubating 1 mM of the corresponding catechol with 2 μM HsaC in I = 0.1 M KPi, pH 7.5 for 10 min. DSHA, DSHBNC, and DSHBNC-CoA each had a molar absorptivity at 392 nm (ε392nm) of 3.8 mM-1 cm-1, determined using an Oxygraph oxygen electrode (Hansatech Instruments Ltd) as described previously [77].   2.2.8 Steady- state kinetic analyses The HsaC and HsaD-catalyzed reactions were followed spectrophotometrically by measuring the increase or decrease in absorbance at 392 nm associated with the production or hydrolysis of the MCP, respectively, using a Varian Cary 5000 spectrophotometer equipped with a thermostatted cuvette holder (Varian Canada, Mississauga, Canada). Assays were performed in 250 µl KPi, pH 7.5 (I = 0.1 M) at 25 ± 0.5 oC. Reactions were initiated by adding HsaD, whose final concentration was such that the progress curves were linear over at least 2 min. Initial velocities were determined from the progress curves using least fit squares as calculated by the kinetics module of the Cary WinUV software. Steady-state parameters were determined by fitting the Michaelis-Menten equation to the data using LEONORA [186]. Buffers were prepared on the day of use. MCPs were prepared and protein was thawed immediately prior to use. The 48  concentration of MCP stock was determined before and after the experiment to correct for non-enzymatic hydrolysis. 2.2.9 KD determination Dissociation constants (KD) for complexes of HsaDMtb S114A with each of DSHA, DSHBNC and DSHBNC-CoA were determined essentially as previously described for DSHA [77]. Briefly, the variant was titrated into 5 μM substrate in an initial volume (Vi) of 200 μl KPi, pH 7.5 (I= 0.1 M) at 25oC and measuring the change in absorbance at 456 nm. The dissociation constant was calculated by plotting the data to the quadratic equation (Equation 1) using the nonlinear curve fitting function of R where Ao = initial absorbance 456 nm, Amax = maximum absorbance 456 nm, [E] = concentration HsaDMtb S114A (μM), and [S] = substrate concentration (μM). Dilution of the substrate was accounted for using Equation 2 where So is the measured initial substrate concentration and VT = the assay volume after each titrant addition. In fitting Equation 1, [S] and [E] were treated as independent variables, and Ao, Amax, and KD, as dependent variables. Equation 1                          ∆ܣସହ଺	௡௠ ൌ 	ܣ௢ ൅	ܣ௠௔௫ሺሺሾாሿାሾௌሿା௄ವሻିඥሺሾாሿାሾௌሿା௄ವሻమିସሾாሿሾௌሿଶሾாሿ  Equation	2																																																																																																							ሾܵሿ ൌ 	 ܵ௢ሺ ௜்ܸܸ ሻ  2.3 Results 2.3.1 Bioinformatic analysis of the steroid catabolic gene cluster in M. abscessus To better characterize the steroid catabolic genes of Mab, a Protein BLAST search was performed for each of 69 proteins from Mtb that have been implicated in cholesterol catabolism in various genomic studies as listed in (Table 2.1) [25, 32, 40, 47]. Orthologs of 65 of these 49  proteins were identified based on being reciprocal best hits, and shared 60-91% amino acid sequence identity with the corresponding protein in Mtb (Table 2.1). The four genes for which orthologs were not found were Rv3519, Rv3528c, FdxB and Rv1106c. The first three of these do not have an experimentally validated role in cholesterol catabolism and none have been predicted to be essential for growth on this sterol [40]. The role of the fourth, a 3β-hydroxysteroid dehydrogenase [70], has been debated due in part to functional redundancy with Rv3502c and Rv3409c, a cholesterol oxidase  [72]. In addition, it is unclear whether Mab possesses an ortholog of Cyp142. The identified best hit, Mab_3825, has 29-34% amino acid sequence identity with four cytochromes P450 (Cyp) from Mtb: Cyp124, Cyp125, Cyp142, and Cyp130. However, Cyp142 appears to be dispensable for growth on cholesterol due to redundancy with Cyp125 [58].  The 65 predicted cholesterol catabolic genes of Mab share significant synteny to those in Mtb, M. smegmatis and RHA1. Moreover, the predicted operonic structure is largely conserved, with the side chain and Rings A/B degradation genes occurring downstream of KstR-binding motifs and the Rings C/D degradation genes occurring downstream of KstR2-binding motifs, as in related bacteria. Nevertheless, the clustering of these genes in Mab appears to be unusual in at least two respects. First, the cholesterol catabolic genes in Mab are arranged in two clusters separated by ~1.39 Mb: the first comprises genes encoding steroid uptake and side chain degradation (Table 2.1; yellow and green, respectively) and the second encoding Rings A/B and C/D degradation (Table 2.1;pink and blue, respectively). In comparison, most of the cholesterol catabolic genes are clustered within 105 kb in Mtb [25], 145 kb in M. smegmatis [187] and 183 kb in RHA1 [25]. A second unusual feature of the gene cluster in Mab concerns the hsaACDB operon, which encodes fragmentation of Rings A/B. Unlike in Mtb, M. smegmatis and RHA1, 50  the operon in Mab lacks hsaD (Figure 2.1A). This gene occurs in a predicted single gene operon, 1.8 Mbp from the hsaACB operon and encodes the only predicted HsaD ortholog in the Mab genome. Interestingly, a mycobacterial KstR-binding site as defined by Kendall et al. [32, 188] occurs 10 bp upstream of the hsaD start codon.  Searching the Mab genome for cholesterol catabolic genes revealed the presence of additional homologs of Rings A/B degradation genes that are not present in Mtb (Table 2.1; Figure 2.1A). Four of these occur in a cluster adjacent to mab_0082c, which encodes a predicted PadR-family transcriptional repressor. Further analyses revealed that these five genes are reciprocal best hits of homologs that specify the catabolism of 4-AD in M. smegmatis [27]. Again, in contrast to the 4-AD catabolic gene cluster of M. smegmatis, the cluster of Mab does not include a homolog of hsaD. However, the Mab genome harbours additional homologs kshB2 and kshA3 (Table 2.1). Finally, no KstR-binding motifs were identified upstream of any these additional steroid catabolic genes.          51  Table 2.1 The steroid and monoaromatic catabolic genes in M. abscessus ATCC 19977 Cholesterolc,dMab gene Gene namea Gene product H37Rv M. smegmatis Identity (%)b MAB_0013ce nat N acetyl-transferase rv3566 MSMEG_0306 32 MAB_0579c kstR TetR transcriptional repressor rv3574c MSMEG_6042 71 MAB_0580 chsE3 acyl-CoA dehydrogenase rv3573c MSMEG_6041 61 MAB_0582c rv3572 hypothetical Protein rv3572 MSMEG_6040 43 MAB_0583c kshB1c ketosteroid hydroxylase, reductase rv3571 MSMEG_6039 67 MAB_0584 hsaA1 3-HSA hydroxylase rv3570c MSMEG_6038 79 MAB_0585 hsaC1 DHSA dioxygenase rv3568c MSMEG_6036 70 MAB_0586 hsaB 3-HSA hydroxylase, reductase rv3567c MSMEG_6035 77 MAB_0593c fadE33 acyl-CoA dehydrogenase rv3564c MSMEG_6016 63 MAB_0594c fadE32 acyl-CoA dehydrogenase rv3563c MSMEG_6015 65 MAB_0595c fadE31 acyl-CoA dehydrogenase rv3562c MSMEG_6014 74 MAB_0596c fadD3 HIP-CoA synthetase rv3561 MSMEG_6013 65 MAB_0597 fadE30 acyl-CoA dehydrogenase rv3560c MSMEG_6012 77 MAB_0598 ipdF short chain dehydrogenase rv3559c MSMEG_6011 82 MAB_0599 kstR2 TetR transcriptional repressor rv3557c MSMEG_6009 71 MAB_0600 fadA6 acyl-CoA thiolase rv3556c MSMEG_6008 79 MAB_0603c ipdC acyl-CoA reductase rv3553 MSMEG_6004 81 MAB_0604c ipdB COCHEA-CoA hydrolase rv3552 MSMEG_6003 74 MAB_0605c ipdA COCHEA-CoA hydrolase rv3551 MSMEG_6002 78 MAB_0606c echA20 HIEC-CoA hydrolase rv3550 MSMEG_6001 78 MAB_0607 rv3549c short chain dehydrogenase rv3549c MSMEG_6000 69 MAB_0608 rv3548c short chain dehydrogenase rv3548c MSMEG_5999 78 MAB_0609c rv3547 hypothetical Protein rv3547 MSMEG_5998 52 MAB_0610  hypothetical Protein - MSMEG_5997 - MAB_0612c fadA5 acyl-CoA thiolase rv3546 MSMEG_5996 76 MAB_0613 cyp125 27-cholesterol oxygenase rv3545c MSMEG_5995 77 MAB_0614 chsE1 acyl-CoA dehydrogenase rv3544c MSMEG_5994 57 MAB_0615 chsE2 acyl-CoA dehydrogenase rv3543c MSMEG_5993 75 MAB_0616 chsH2 MaoC-like enoyl-CoA hydratase rv3542c MSMEG_5992 71 MAB_0617 chsH1 MaoC-like enoyl-CoA hydratase rv3541c MSMEG_5991 75 MAB_0618 ltp2 lipid Transfer Protein rv3540c MSMEG_5990 83 MAB_0621c hsd4B hydroxyl steroid dehydrogenase rv3538 MSMEG_5943 62 MAB_0622c kstD1 ketosteroid dehydrogenase rv3537c MSMEG_5941 78 MAB_0624 hsaE hydratase rv3536c MSMEG_5940 71 MAB_0625 hsaG acetaldehyde dehydrogenase rv3535c MSMEG_5939 87 MAB_0626 hsaF 4-hydroxy-2-oxovalerate aldolase rv3534c MSMEG_5937 91 MAB_0685e aspB aminotransferase rv3565 MSMEG_6017 59 MAB_0718ce rv0139 oxidoreductase rv0139 MSMEG_6474 29 MAB_3719c choD cholesterol oxidase rv3409c MSMEG_1604 79 MAB_3810e hsaD DSHA hydrolase rv3569c MSMEG_6037 36 - f cyp142 cytochrome P450 rv3518c MSMEG_5918  MAB_4148c mce4F ABC transporter rv3494c MSMEG_5895 65 MAB_4149c mce4E ABC transporter rv3495c MSMEG_5896 57 MAB_4150c mce4D ABC transporter rv3496c MSMEG_5897 58 MAB_4151c mce4C ABC transporter rv3497c MSMEG_5898 63 MAB_4152c mce4B ABC transporter rv3498c MSMEG_5899 66 MAB_4153c mce4A ABC transporter rv3499c MSMEG_5900 58 MAB_4154c yrbE4B ABC transporter rv3500c MSMEG_5901 78 MAB_4155c yrbE4A ABC transporter rv3501c MSMEG_5902 87 MAB_4156c hsd4A hydroxyl steroid dehydrogenase rv3502c MSMEG_5903 72 MAB_4157c fdxD Ferredoxin rv3503c MSMEG_5904 70 MAB_4158 chsE4 acyl-CoA dehydrogenase rv3504 MSMEG_5906 84 MAB_4159 chsE5 acyl-CoA dehydrogenase rv3505 MSMEG_5907 65 MAB_4160 fadD17 acyl-CoA synthetase rv3506 MSMEG_5908 60 MAB_4162c MSMEG_5913 nitronate monooxygenase - MSMEG_5913 - MAB_4163c fadD19 27-cholestenoyl-CoA synthetase rv3515c MSMEG_5914 68 MAB_4164 echA19 enoyl-CoA hydratase rv3516 MSMEG_5915 79 MAB_4166c Rv3520c luciferase-like Protein rv3520c MSMEG_5920 78 MAB_4167 Rv3521 hypothetical Protein rv3521 MSMEG_5921 73 MAB_4168 ltp4 lipid Transfer Protein rv3522 MSMEG_5922 77 MAB_4169 ltp3 lipid Transfer Protein rv3523 MSMEG_5923 87 52  Cholesterol (cont.) Mab gene Gene namea Gene product H37Rv M. smegmatis Identity (%)b MAB_4173 kshA1 ketosteroid hydroxylase, oxygenase rv3526 MSMEG_5925 75 MAB_4174 rv3527 hypothetical Protein rv3527 MSMEG_5927 53 MAB_4177c rv3529c hypothetical Protein rv3529c MSMEG_5930 72 MAB_4178c rv3530c hypothetical Protein rv3530c MSMEG_5931 68 MAB_4179c rv3531c hypothetical Protein rv3531c MSMEG_5932 69 4-AD catabolismMab gene Gene namea Gene product H37Rv M. smegmatis Identity (%)b - hsaD2 DSHA hydrolase - MSMEG_2900 - - kstD3 ketosteroid dehydrogenase - MSMEG_2867 - MAB_0075 hsaA2 3-HSA hydroxylase, oxygenase - MSMEG_2892 77MAB_0076 hsaC2 DHSA dioxygenase - MSMEG_2891 75MAB_0080c kshA2 ketosteroid hydroxylase, oxygenase - MSMEG_2870 75MAB_0081c kstD2 ketosteroid dehydrogenase - MSMEG_2869 66MAB_0082c MSMEG_2868 PadR-like transcriptional repressor - MSMEG_2868 52MAB_3627c kshA3 ketosteroid hydroxylase, oxygenase - - - MAB_3918 kshB2 ketosteroid hydroxylase, reductase - MSMEG_2893 57Isopropyl benzeneMab gene Gene name Gene product R. erythropolis BD2 Identity (%)g MAB_4358c - 2,3-dihydroxybiphenyl 1,2 dioxygenase III - - MAB_4361 mphD 2-keto-4-pentenoate hydratase - - MAB_4365c ipbB IPB dihydrodiol dehydrogenase PBD2.158 62 MAB_4366c ipbD HOMODA hydrolase PBD2.174 50 MAB_4367c ipbA2 IPB dioxygenase, small subunit PBD2.154 60 MAB_4368c ipbA4 IPB dioxygenase, ferredoxin reductase PBD2.156 49 MAB_4369c ipbA3 IPB dioxygenase, ferredoxin PBD2.155 50 MAB_4370c ipbA1 IPB dioxygenase, large subunit PBD2.153 54 MAB_4372 - 3-(2,3-dihydroxyphenyl) propionic acid dioxygenase - -       aIn the absence of a gene name, the H37Rv (cholesterol) or M. smegmatis (4-AD) gene ID is listed. bFor cholesterol catabolic gene products, the indicated amino acid sequence identity is between M. abscessus and M. tuberculosis proteins. For 4-AD catabolic gene products, the value is between M. abscessus and M. smegmatis proteins. cGenes in purple, green, red and blue font are involved in cholesterol uptake, side chain degradation, Rings A/B degradation and Rings C/D degradation, respectively.  d No reciprocal best hit for hsd (rv1106c),  fadD18 (rv3513c), rv3519, rv3528c, and fdxB (rv3554), predicted to be involved in cholesterol catabolism in M. tuberculosis, were identified in M. abscessus  eReciprocal best hit between M. abscessus ATCC 19977 and M. tuberculosis H37Rv fMab_3825 occurs in this cluster but is not the reciprocal best hit of Cyp142 from M. tuberculosis, sharing  34%, 33%, 33%, and 29% amino acid identity with M. tuberculosis Cyp124, Cyp125, Cyp142 and Cyp130, respectively. gCLUSTAL alignment (BLOSM62) identity displayed.  2.3.2 M. abscessus possesses a HsaD ortholog dissimilar from other actinobacteria In light of the finding that hsaDMab does not cluster with known steroid catabolic genes, phylogenetic analyses were preformed to better understand the gene product’s relationship with other HsaDs. Previous analyses had identified three Subfamilies (I to III) comprising enzymes involved in the catabolism of biphenyl/steroids, alkylated benzenes and dibenzofuran-like compounds, respectively [189]. By contrast, the updated phylogenetic analyses performed using a structure-based alignment and a larger number of sequences identified seven subfamilies of MCP hydrolases (Figure 2.1B). The enzymes appear to group largely according to their substrate 53  specificities, as seen previously [189]. However, the newly defined Subfamily I comprises only biphenyl-degrading homologs and three distinct subfamilies of steroid-degrading MCP hydrolases were identified: Subfamily IV, comprising cholesterol catabolic enzymes from Actinobacteria; Subfamily V, cholate and 4-AD catabolic enzymes from Actinobacteria; and Subfamily VI, cholate and testosterone catabolic enzymes from Gram negative strains. Subfamily VII includes enzymes involved in the degradation of 3-(hydroxyphenyl)propionate and related compounds.  HsaDMab belonged to Subfamily V which includes the cholate-degrading HsaD3 from RHA1 [22, 93] as well as HsaD2 from RHA1 and HsaD2 from M. smegmatis, both of which are involved in 4-AD catabolism [22, 190]. These enzymes are not predicted to act on steroids with partially degraded side chains [93, 190], in contrast to the cholesterol-catabolising HsaDRHA1 and HsaDMtb.  A second MCP hydrolase, Mab_4366c, is encoded within the Mab genome and displays 36% amino acid sequence identity (88% coverage) with HsaDMtb. Although the phylogenetic analysis indicated that it occurs within Subfamily IV (Figure 2.1), the bioinformatics evidence indicates that it is unlikely to act on a steroid-derived substrate. The mab_4366c gene does not occur adjacent to known steroid catabolic genes nor is a KstR motif located upstream. More specifically, mab_4366c occurs within a predicted six-gene operon, mab_4365c - mab_4370c, encoding enzymes predicted to be involved in isopropyl benzene (Ipb) degradation (Table 2.1) [191]. A similarly organized Ipb cluster was characterized in Rhodococcus erythropolis BD2 whose gene products are the reciprocal best hits of the M. abscessus genome are located within this cluster sharing 35 – 45% amino acid identity (Table 2.1) [192, 193]. Therefore, Mab_4366c 54  is predicted to be involved in isopropyl benzene catabolism and is provisionally annotated as IpdD.  Figure 2.1 The steroid catabolic gene clusters in M. abscessus (A) Organization of actinobacterial Rings A and B degrading enzymes involved in cholesterol (red), ADD (blue), cholate (green), and undetermined (grey) steroid catabolism. Genes are identified above by their respective gene product and below by their loci. Omitted are mab_, rv, msmeg_, and RHA1_RS prior to the gene loci for M. abscessus, M. tuberculosis, M. smegmatis, and R. jostii RHA1. (B) Phylogenetic analysis of MCP hydrolases. The tree was generated using a structure-based alignment of sequences the following MCP hydrolases: HOPDA hydrolase from Bacillus sp. Strain JF8 (BphDJF8, BAC79225); HOPDA hydrolase from Burkholderia xenovorans LB400 (BphDLB400, WP011494293.1); HOPDA hydrolase from E. coli (MhpC, BAA13054.1); 2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoate hydrolase from sphingomonas sp. strain KA1 (CarC, BAC56762); 6-isopropyl-HOHD hydrolase from Pseudomonas putida F1 (CmtE, BAB17778); MCP hydrolase from Pseudomonas sp. strain 55  CF600 (DmpD, P19076); chloro-HOPDA hydrolase from Sphingomonas wittichii RW1 (DxnB2, WP012049251.1); HOHD hydrolase from R. jostii RHA1 (IpbD, BAA31163); HOHD hydrolase from Pseudomonas fluorenscens (EtbD1, BAA12150); DSHA hydrolase from M. avium (HsaDMav, WP033725372.1); DSHA hydrolase from M. intracellulare (HsaDMin, WP014381744.1); DSHA hydrolase from M. smegmatis (HsaDMsm, AFP42309.1); DSHA hydrolase from M. tuberculosis (HsaDMtb, NP_218086.1); DSHA hydrolase from M. ulcerans (HsaDMul, WP011741784.1); DSHA hydrolase from Rhodococcus rhodochrous (HsaDRrh, WP019746906.1); DSHA hydrolase from R. jostii RHA1 (HsaDRHA1, BAA98136); DSHA hydrolase from M. smegmatis (HsaD2Msm, AFP39291.1); MCP hydrolase from R. rhodochrous (HsaD2Rrh, WP085469424.1); TDSHA hydrolase from R. jostii RHA1 (HsaD3RHA1, ABG97574); MCP hydrolase from R. jostii RHA1 (HsaD4RHA1, ABH00058); DSHA from M. abscessus (HsaDMab, Mab_3810); 2-hydroxymuconic-semialdehyde hydrolase from R. jostii RHA1 (OhpC, ABG92355); HOPDA hydrolase from Pseudomonas sp. strain DJ12 (PcbD, BAA07955); 2-hydroxymuconic-semialdehyde hydrolase from R. jostii RHA1 (PheC, ABG99128); TDSHA hydrolase from Comamonas testosteroni TA441 (TesDTA441, BAC67693); TDSHA hydrolase from Pseudomonas putida Chol1 (HsaDChol1; WP008568663.1); 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase from Pseudomonas putida F1 (TodF, P23133); and putative HOHD hydrolase from M. abscessus (Mab_4366c, CAM64437).  2.3.3 M. abscessus grows on cholesterol and 4-AD but not cholate The identification of multiple clusters of steroid catabolic genes in Mab prompted investigations into the ability of the bacterium to grow on different steroids. The strain grew to stationary phase within 72 h on cholesterol with a doubling time of approximately 12 hours, and a concurrent depletion of cholesterol from the media was observed by GC-MS (Figure 2.2A). Mab also grew on 4-AD, although to a lower yield, commensurate with the steroid’s lack of a side chain (Figure 2.2B). Further, 4-AD-grown cultures turned pink, consistent with the accumulation and non-enzymatic oxidation of DHSA, a catechol [39] (Figure 2.2D). GC-MS analysis confirmed the presence of DHSA based on its molecular ion (m/z = 460) and fragmentation pattern (Figure 2.2E). The accumulation of DHSA could reflect the inefficient expression of hsaD in the presence of 4-AD due to repression by KstR leading to upstream metabolite accumulation. Finally, Mab did not grow on cholate or detectably deplete cholate from the media (Figure 2.2C), consistent with bacterium’s predicted lack of cholate catabolic genes.   56   Figure 2.2 Growth of M. abscessus on different steroids. M. abscessus was grown in M9 minimal medium supplemented with 0.5 mM cholesterol (A), 0.5 mM 4-AD (B), or 0.5 mM cholate (C). Growth was measured by optical density at 600 nm (solid lines), and substrate depletion was measured by GC-MS (dotted lines). Error bars indicate standard deviation of biological triplicates. (D) Colour of culture supernatants of M. absceussus grown on cholesterol and 4-AD. (E) GC-MS acquired mass spectra of DHSA identified in culture supernatant of ADD grown M. absceussus.  2.3.4 ΔhsaC and ΔhsaD RHA1 accumulate metabolites with incompletely degraded sidechains. The data imply that HsaDMab is involved in growth on cholesterol. However, the phylogenetic data indicate that the enzyme’s substrate specificity (kcat/KM) differs from that of other HsaDs involved in cholesterol catabolism, such as HsaDMtb and HsaDRHA1. To probe these differences, metabolite accumulation in the previously described hsaC and hsaD mutants of RHA1 [25] was first investigated. RHA1 is used to understand cholesterol catabolism and its mutants are used to generate metabolites to study Mtb enzymes [58, 76]. When incubated with cholesterol, an hsaC mutant accumulated two metabolites that were detected using GC-MS and that were not detected in cultures of WT cells (Figure 2.3, red). The first was DHSA, as 57  previously observed for this mutant [39]. The second had a parent ion with m/z = 590 and contained the 294 m/z fragment characteristic of the catechol of DHSA (Figure 2.3, red). NMR analysis identified this metabolite as 3,4-dihydroxy-17-isopropionoyl-9,10-seconandrost-1,3,5(10)-triene-9-one (DHSBNC), a DHSA analog with a C-17 isopropionyl moiety (data not shown). Cultures of ΔhsaD cells incubated with cholesterol were characterized by yellow-colored supernatants (data not shown). This coloration is consistent with excretion of an MCP such as DSHA [77, 159]. GC-MS analysis identified a metabolite in these supernatants whose parent ion had m/z = 549 (Figure 2.3, blue). This value is consistent with the 2-amino derivative of DSHBNC (DSHA with a C-17 isopropionyl group). MCPs have been reported to react with ammonia forming pyridine analogs [194]. These results are consistent with the mutant excreting DSHBNC which then reacts with ammonium chloride, present at ~20 mM in M9 media, to yield 2-amino DSHBNC. No metabolites with masses consistent with DSHA or 2-amino-DSHA were detected, implying that DSHBNC is the primary metabolite that is excreted in ΔhsaD RHA1. 58   Figure 2.3 ΔhsaC and ΔhsaD RHA1 accumulate cholesterol derived metabolites with partially degraded side chains. Shown are gas chromatograms of ethyl acetate extracted supernatants from ΔhsaC (red), ΔhsaD (blue), and WT RHA1 following 48 h of incubation in M9G containing 0.5 mM cholesterol. Structures displayed represent species confirmed using authentic standards.  2.3.5 The substrate specificities of HsaCMtb, HsaDMab and HsaDMtb. The accumulation of DHSBNC in the culture supernatant of ΔhsaC RHA1 indicates that HsaC may act on this substrate or a CoA thioester, thereof. DHSBNC-CoA was produced from DHSBNC using the CoA synthetase CasI. Steady state kinetic analysis of HsaCMtb revealed a ~4 times higher substrate specificity for DHSBNC-CoA than either DHSA or DHSBNC (Figure 2.4, Table 2.2).   To further characterize HsaDMab and HsaDMtb, their respective substrate specificities were compared. Both enzymes had higher specificity (kcat/KM) for a CoA thioesterified substrate, DSHBNC-CoA, than for non-thioesterified substrates, DSHA and DSHBNC (Figure 2.4, Table 59  2.2). However, the difference was two orders of magnitude in HsaDMtb and only a single order of magnitude in HsaDMab. This effect can largely be attributed to the significantly lower KM value for DSHBNC-CoA as compared DSHA or DSHBNC for either enzyme. Despite both enzyme’s higher specificity for DSHBNC-CoA, HsaDMab nevertheless had a reasonable kcat/KM value for DHSA, the 4-AD metabolite, ~40-fold higher than that of HsaDMtb. Finally, HsaDMtb had remarkably similar-steady state kinetic parameters for DSHA and DSHBNC, suggesting that the C-17 isopropionyl group contributes little to the substrate specificity. In an attempt to determine the affinity of HsaDMtb for the CoA moiety, competitive inhibition of the DSHBNC-CoA reaction was tested using CoASH but no significant inhibition was detected (Ki,c > 2 mM, data not shown).   Figure 2.4 Steady-state kinetic parameters of HsaCMtb and MCP hydrolases towards substrates with and without partially degraded side chains Shown are steady state analyses of (A) HsaCMab using DHSBNC (blue) or DHSBNC-CoA (black) and (B) HsaDMab using DSHBNC (blue) or DSHBNC-CoA (black) as substrates. Rates reported are normalized to enzyme concentration. Reactions were performed in 200 μl potassium phosphate pH 7.5 (I = 0.1 M) at 25oC and followed spectrophotometrically at 392 nm. Curves represent best fit of the Michaelis-Menten equation.          60    Table 2.2 Steady-state kinetic parameters of HsaCMtb, HsaDMtb and HsaDMtb  Enzyme Substrate kcat (s-1) KM (μM) kcat  /  KM (x 105 M-1 s-1)HsaCMtb DHSA 8.0 (0.2) 1.6 (0.1) 50 (4) DHSBNC 6.6 (0.1) 3.2 (0.3) 21 (2) DHSBNC-CoA 13.4 (0.3) 0.63 (0.06) 210 (30) HsaDMtb DSHA 0.93 (0.04) 34 (3) 0.27 (0.02) DSHBNC 0.91 (0.04) 35 (3) 0.26 (0.02) DSHBNC-CoA 3.4 (0.1) 0.6 (0.2) 57 (3) HsaDMab DSHA 9 (1) 8(2) 11 (1) DSHBNC 6.7 (0.3) 3.2 (0.4) 21 (2) DSHBNC-CoA 9.5 (0.5) 0.5 (0.1) 180 (20)  2.3.5.1 HsaDMtb has high affinity for substrates with partially degraded side chains Steady-state kinetic analysis established that HsaDMtb has >200 times higher specificity for substrates with partially degraded side chains. This effect is largely attributed to the presence of the CoA moiety in DSHBNC-CoA (Table 2.2). We speculated that this effect is reflective of tight binding between DSHBNC-CoA and HsaDMtb. The affinity (KD) of HsaDMtb for substrates with a partially (DSHBNC-CoA, DSHBNC) and completely degraded (DSHA) side chain was determined by titrating solutions of substrate with the catalytically inactive variant HsaDMtb S114A.  The HsaDMtb S114A·MCP complex has a characteristic orange color whose absorption spectrum is similar to that of the catalytic intermediate, ESred [77]. Titration of HsaDMtb S114A into DSHBNC-CoA resulted in the same bathochromic shift of the substrate from a λmax of 392 nm to 459 nm (Figure 2.5, inset). HsaDMtb S114A had over 500-times greater affinity for 61  DSHBNC-CoA than for either DSHA or DSHBNC (Figure 2.4;Table 2.3). The similar affinity of HsaDMtb S114A for DSHA and DSHBNC is consistent with HsaDMtb’s similar specificity for these substrates. The quadratic binding equation calculated a lower Amax for DSHA and DSHBNC/DHSBNC-CoA, suggesting a smaller extinction coefficient of ESred for the HsaDMtb S114A·DSHA complex than with the other two substrates (Table 2.3). Overall, the presence of the CoA moiety could contribute and additional ~16 kJ mol-1 of binding energy of DSHBNC-CoA over DSHBNC which may be responsible for the higher substrate specificity of HsaDMtb towards DSHBNC-CoA.  Figure 2.5 Dissociation constant (Kd) determination for HsaDMtb towards DSHBNC-CoA. Shown is the increase in absorbance at 459 nm following the titration of HsaD from M. tuberculosis into 5 μM DSHA (red), DSHBNC (blue) or DSHBNC-CoA (black). Spectra were recorded in 200 μl potassium phosphate pH 7.5 (I =0.1M) at 25oC. Curves represent the best fit of the quadratic binding equation to the data. Inset displays the change in spectra of DSHBNC-CoA with (dotted line) and without (solid line) HsaD from M. tuberculosis..   62  Table 2.3 Parameters for dissociation constant determination of HsaDMtb Substrate Ao (units) Amax (units) KD (μM) DSHA 0.002 (0.001) 0.066 (0.004) 34 (6) DSHBNC 0.003 (0.001) 0.094 (0.003) 32 (4) DSHBNC-CoA 0.0014 (0.0007) 0.095 (0.001) 0.06 (0.02)   63   Structural and functional characterization of the KstR2∙HIP-CoA Chapter 3:complex 3.1 Introduction. HIP, the metabolite resulting from the degradation of cholesterol’s alkyl side chain and Rings A and B, is a major metabolic check point for the degradation of the final steroid fragment. Casabon et al. (2013) demonstrated that the degradation of HIP commences by its thioesterification to HIP-CoA. Therefore, subsequent steps in its catabolism were proposed to involve CoA thioesters. Interestingly, HIP-CoA regulates the expression of a ~14-gene regulon by acting as the effector of KstR2, the regulon’s TetR family transcriptional repressor [91]. However the mechanism by which HIP-CoA acts on KstR2 is unknown. Herein, a combination of isothermal titration calorimetry (ITC), electrophoretic mobility assays (EMSA), X-ray crystallography and directed mutagenesis was used to characterize the molecular function of KstR2 from M. tuberculosis, KstR2Mtb. The data define interactions between KstR2 and HIP-CoA, and provide insights into the function of this regulator in the bacterial catabolism of steroids as well as into TFRs in general.  The contents of Chapter 3 were published in the Journal of Biological Chemistry in 2015 [177]. Experiments pertaining to the crystallization and structure refinement were all performed by Dr. Peter Stogios and Elena Evdokimova in the Savchenko Lab at the University of Toronto. Sections 3.2.5 and 3.3.2 were written primarily by PS. All other sections were written and edited by LE and myself. 64  3.2 Materials and methods 3.2.1 Chemicals and reagents ATP, CoASH, and cholesterol (>99%) were purchased from Sigma-Aldrich. NdeI and HindIII Fast Digest restriction enzymes were purchased from Thermo Fisher Scientific Inc. T7 DNA Ligase and DpnI were purchased from New England Biolabs. Oligonucleotides were ordered from Integrated DNA Technologies. FadD3 and poly-His-tagged tobacco etch virus protease (TEVPro) were produced as previously described [79, 195]. Water for buffers was purified using a Barnstead Nanopure DiamondTM system to a resistivity of at least 18 MΩ. Reagents were of HPLC or analytical grade. 3.2.1.1 Preparation of HIP and HIP-CoA HIP was obtained using a ΔfadD3 mutant of RHA1 as previously described [91]. HIP-CoA was produced by incubating 2 mM HIP with 2.25 mM ATP, 2.25 mM CoASH, 5 mM MgCl2 and 5 μM FadD3 in 800 μl 25 mM HEPES, pH 7.5, 50 mM KCl for 30 min. HIP-CoA was purified at room temperature by high-performance liquid chromatography (HPLC) using a Luna 3 μm PFP(2) 50 × 4.6 mm column (Phenomenex) in 100 mM ammonium acetate, pH 4.5 at 1 ml min-1 over a 20 ml linear gradient of 0-90% methanol. HIP-CoA containing fractions were pooled and methanol was removed under nitrogen. HIP-CoA was purified to >95% purity and its identity was confirmed by ESI-MS as described [91]. HPLC purified HIP-CoA was desalted using a Strata-X 33u 30 mg solid phase extraction (SPE) column (Phenomenex). The SPE column was equilibrated with 1 ml methanol then 1 ml water. The HIP-CoA solution was passed through the column, washed with 1 ml water, and eluted in 100% methanol. HIP-CoA was dried under nitrogen and solubilized in 100 μl 25 mM HEPES, pH 7.5, 50 mM KCl. Its concentration was determined spectrophotometrically using ε260 nm = 11.9 mM-1 cm-1. Typical mole recoveries 65  ranged from 70-80%. HIP-CoA for co-crystallization was produced as described above but in a final volume of 6 ml (2.9 mg HIP). HPLC-eluted fractions containing high concentrations of HIP-CoA were desalted on the SPE column, dried under nitrogen, suspended in 250 µl water and lyophilized overnight. The residue was suspended in 50 µl water to a final concentration of 62 mM. 3.2.2 DNA manipulation Plasmid DNA was manipulated and propagated using standard procedures [180]. Oligonucleotide-directed mutagenesis was performed using the QuickchangeTM PCR protocol with slight modifications. Briefly, a single 5’ phosphorylated mutagenic DNA primer was annealed to pETKstR2 carrying a gene encoding poly-His tagged (Ht-)KstR2Mtb  [91], then amplified using Phusion DNA Polymerase. T7 DNA ligase was added to the reaction mixture to form single stranded mutagenized plasmid DNA. Template DNA was removed using DpnI and the remaining ssDNA was electroporated into Escherichia coli NovaBlue. The R162M and W166L variants were producing using primers with the following respective nucleotide sequences: 5’-pGTCTACCGATTCATCATGGACACCACCTGGGTG-3’ and 5’-pCATCCGTGACACCACCCTCGTGTCGGTGCGCTGG-3’. The nucleotide sequences of variant kstR2 were confirmed. 3.2.3 Purification of KstR2 and variants Wild-type and variant KstR2Mtb were produced using E. coli Rosetta 2(DE3)pLysS carrying the appropriate derivative of pETKstR2 as previously described [91]. The proteins were purified as previously described [91] with the following modification. The affinity-purified Ht-KstR2Mtb was dialyzed overnight against cleavage buffer (25 mM HEPES, pH 7.5, 50 mM KCl, 1 mM DTT, and 0.5 mM EDTA). The affinity tag was removed by incubating ~100 mg of Ht-KstR2Mtb 66  with 0.5 mg TEVPro in 10 ml cleavage buffer overnight at 4oC. Complete digestion was confirmed by SDS-PAGE analysis. TEVPro-digested KstR2Mtb was loaded onto Mono-Q 10/100 HR (GE Healthcare) and eluted as previously described [91]. Proteins were exchanged into 25 mM HEPES, pH 7.5, 50 mM KCl, concentrated to ~20 mg ml-1 and flash frozen in liquid nitrogen as beads. Typically, 50 mg of protein were purified per 1 l culture.  Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay with bovine serum albumin as a standard. 3.2.4 Functional characterization of KstR2 3.2.4.1 Isothermal titration calorimetry ITC experiments were performed using an ITC200 instrument (GE Healthcare) operated at 25oC and a stirring speed of 1000 rpm. Titrations were performed using 25 mM HEPES, pH 7.5, 50 mM KCl. KstR2Mtb (20 μM or 40 μM variant) was titrated with 40 × 1 μl injections of HIP-CoA (200 or 400 μM). For HIP and CoASH, 40 μM KstR2Mtb was titrated with 20 × 4 μl injections of 400 μM titrant. Injections of buffer into KstR2Mtb and variants showed no significant background heats. The data were processed by subtracting the background heats and removing outlier data points. One- and two-site models were fit using Origin 7.0. Experiments were independently repeated at least three times. 3.2.4.2 Electrophoretic mobility shift assays A dsDNA probe of the KstR2Mtb operator sequence located in the intergenic region of rv3557c and rv3558 was prepared by heating complementary ssDNA oligomers to 95oC and annealing at room temperature in 20 mM Tris-HCl, pH 8.0, 10 mM MgCl2, and 75 mM NaCl as previously described [91]. DNA probes were labelled with DIG-11-ddUTP using the second generation DIG gel shift kit from Roche according to the manufacturer’s protocol. Binding 67  assays contained 0-2 pmol KstR2Mtb (WT or variant), 0.04 pmol DIG-labelled DNA probe and 0-10 nmol HIP-CoA in 20 μl 20 mM HEPES, pH 7.6, 10 mM (NH4)2SO4, 1 mM DTT, 0.2% (w/v) Tween 20, 30 mM KCl, and 1 mM EDTA. Assays were incubated for 30 min at 37oC then loaded onto 9% polyacrylamide gels containing 0.5× TBE. Gels were run for 45 min at 105 V then blotted onto positively charged Hybond-N+ nylon membranes (GE Healthcare). DNA was viewed using anti-DIG-alkaline phosphatase and chemi-luminescent substrate, CSPD, as described by the manufacturer (Roche). Sequences of DNA probes were 5’-GCGTACCAAGCAAGTGCTTGCTTAGGTAGC-3’ and 5’-GCTACCTAAG-CAAGCACTTGCTTGGTACGC-3’. 3.2.4.3 Size exclusion chromatography The oligomeric state of KstR2 was analyzed using size exclusion chromatography multi-angular light scattering (SEC-MALS). Twenty five μl of 80 μM KstR2Mtb was injected onto a HPLC 1260 Infinity LC (Agilent Technologies) coupled to a Superdex 200 5/150 column (GE Healthcare). A second sample was incubated at 37oC for 30 min with 20 μM of a 24-bp DNA fragment representing the KstR2 operator from the intergenic region of rv3549c/rv3550 [83]. SEC-MALS was operated at 0.2 ml min-1 in 25 mM HEPES, 50 mM KCl, pH 7.5. Data were collected using a miniDAWN TREOS multi-angle static light scattering device and an Optilab T-rEX refractive index detector (Wyatt Technologies). The molecular weight of complexes was determined using the ASTRA6 program (Wyatt Technologies). The nucleotide sequences of the oligonucleotides used to generate the DNA fragment were 5’-ACCTAAGCAAGC-ACTTGCTTGGTA-3’ and its complement. Oligo-nucleotides were HPLC-purified by the manufacturer (Integrated DNA Technologies) and annealed as described above in 25 mM HEPES, 50 mM KCl, pH 7.5. 68  3.2.5 Crystallization of KstR2Mtb:HIP-CoA Crystals of the KstR2Mtb·HIP-CoA complex were obtained by mixing 2 μl of 48 mg ml-1 protein with HIP-CoA at final concentration of 1 mM and 2 μl of reservoir solution (0.2 M ammonium sulfate, 0.1 M bis-Tris, pH 5.5 and 25% (w/v) PEG3350) using the hanging drop vapor diffusion method. Crystals appeared at room temperature and were flash frozen in liquid nitrogen after being cryoprotected with paratone oil.  X-ray diffraction data was collected at 100 K using a Rigaku HomeLab system featuring Micromax-007 HF rotating copper anode fitted with a Rigaku R-AXIS IV++ image plate detector.  Diffraction data was processed and reduced using the HKL-3000 software package [196]. The crystal structure was solved by molecular replacement using MrBump from the CCP4 software package [197] with the structure of KstR2RHA1 (Ro04598) from R. jostii RHA1 (PDB 2IBD) as a search query.  Structure refinement was carried out using Phenix.refine [198] and Coot [199].  Geometry was verified using Phenix.refine, Coot and the RCSB PDB Validation server. The final asymmetric unit (AU) contains one copy of the KstR2Mtb protein chain encompassing residues 3-198. The presence of one copy of HIP-CoA in the AU was verified using simulated annealing (Cartesian) omit maps using Phenix.refine with default parameters, followed by model building into residual positive Fo-Fc density and occupancy refinement of HIP-CoA 3.2.5.1 Structural analysis The PDBePISA server was used to analyze inter-protomeric and protein-ligand interactions [200]. The DaliLite and PDBeFold servers were utilized for structure comparisons [201, 202]. Electrostatic surfaces were analyzed using Chimera [203]. Binding cavity properties were analyzed using the CASTp server [204]. 69  3.3 Results 3.3.1 KstR2Mtb binds HIP-CoA with high affinity HIP-CoA is the chemical effector of KstR2Mtb, relieving the binding of the repressor to its operator DNA upon interaction with this molecule [91].  ITC was performed to better analyze the interaction of KstR2Mtb with its effector. The binding was exothermic and driven by enthalpy with an unfavorable entropic contribution (Figure 3.1,Table 3.1). The one-site equation best fit the binding isotherms. No cooperativity was detected and attempts to model the data using a two-site equation yielded poor fits. Replicate titration curves were centered at a mole ratio of 0.89 consistent with a one-to-one stoichiometry between the KstR2Mtb protomer and HIP-CoA. Under the experimental conditions, the Kd was 80 ± 10 nM (25 mM HEPES, pH 7.5, 50 mM KCl).  ITC was further employed to test whether KstR2Mtb binds either HIP or CoASH. Titrations of 400 μM HIP or CoASH into 40 μM KstR2Mtb (Figure 3.1 B and C, respectively) gave heats that were slightly above background. However, neither compound yielded a titration curve. Increasing the concentrations to 1 mM titrant and 100 μM KstR2Mtb generated a proportional increase in measured heats for both compounds and no titration of CoASH. Titrations at 1 mM HIP were unreliable due to precipitation of KstR2Mtb during titration. Table 3.1 Thermodynamic parameters of KstR2Mtb binding HIP-CoA. KstR2 variant Ligand N Kd (μM) ΔH (kJ mol-1) ΔS (J K-1mol-1) WT HIP NBa - - - WT CoASH NB - - - WT HIP-CoA 0.89 (0.01) 0.08 (0.01) -69.4 (0.5) -56 R162M HIP-CoA 1.77 (0.02) 16 (2) -43.0 (0.9) -66 W166L HIP-CoA NB - - - aNo binding detected. 70   Figure 3.1 Representative isotherms of potential KstR2Mtb ligands. (A). Titration of 20 μM KstR2Mtb with aliquots of 200 μM HIP-CoA. (B). Corresponding isotherm with the best fit single binding site model. (C). Titration of 40 μM KstR2Mtb with aliquots of 400 μM CoASH. (D). Titration of 40 μM KstR2Mtb with aliquots of 400 μM HIP. Titrations were performed using 25 mM HEPES 50 mM KCl, pH 7.5 at 25oC.  3.3.2 Structure of KstR2Mtb:HIP-CoA reveals an effector binding cleft spanning the two protomers of the dimer  To further understand the molecular function of KstR2Mtb, KstR2Mtb was crystallized in the presence of HIP-CoA and the structure of the complex was solved to 1.6 Å. The asymmetric unit of the KstR2Mtb·HIP-CoA structure contained a single protomer of KstR2Mtb associated with one molecule of HIP-CoA (Figure 3.2) in agreement with our ITC analysis. Structure determination statistics are presented in Appendix B. 71  Similarly to previously characterized TFRs, the KstR2Mtb protomer adopted an all α-helical L-shaped fold covering approximately 25 × 38 × 57 Å (Figure 3.2A). The short axis of the protomer comprises the N-terminal DBD (residues 6 to 54) with most α-helices in this domain arranged perpendicular to the long axis of the protein representing the C-terminal EBD (residues 55-198) (Figure 3.2A). The two domains are connected by a kinked α-helix (α6) with one face of this helix interacting with the DBD and the other with the EBD.  The KstR2Mtb protomer formed an extended interface of 1687 Å2 with an adjacent KstR2Mtb protomer related to the first by a crystallographic two-fold symmetry axis. This arrangement likely represents the biological dimer, the typical minimal oligomeric state of TFRs. Dimerization of KstR2Mtb is mediated by contacts between 24 residues, 15 of which are hydrophobic, belonging to α-helices α8 and α9 of the C-terminal domain of each protomer. The electron density corresponding to the HIP-CoA molecule (Figure 3.2B) occupies a large extended cavity (2637 Å2 in surface area) that spans the two KstR2Mtb protomers and that sequesters over half of the ligand molecule from the solvent. This cavity, whose shape and chemical nature closely complement that of the ligand, is composed of two elements: a positively charged pocket lined by helices α8, α9, and their connecting loop in one protomer that binds the adenosine moiety; and a deep hydrophobic pocket defined by helices α4’, α5’, α6’, α7’ and α8’ in the second protomer that binds the HIP moiety (’ identifies elements of the second protomer).  Thus, each HIP-CoA molecule binds across the KstR2Mtb dimer interface (Figure 3.2), and the two binding clefts are independent of each other. Indeed, the two ligands approach no closer than 7.6 Å. A total of 17 amino acids from each KstR2Mtb protomer are located within 4.0 Å and HIP-CoA and interact with the HIP-CoA molecule. The adenine moiety of the ligand is anchored 72  primarily through interactions with residues belonging to the loop connecting helices α8 and α9.  The diphosphates of the CoA moiety are stabilized by four hydrogen bonds, including three with Arg-162’. The cycloalkanone rings of the HIP moiety form stacking interactions with the aromatic side chains of Trp-166’ (from α8’) and Tyr-108’ (from α6’) that make up the deep hydrophobic pocket. In addition, the HIP moiety forms many hydrophobic interactions with side chains of residues that line the pocket: Phe-65’, Leu-66’, Leu-69’, Phe-70’, Tyr-73’ and Val-105’. Finally, the 5-carbonyl oxygen forms a hydrogen bond with the side chain of Gln-109’. The high number of protein-ligand contacts and the close complementarity between the chemical environment of KstR2Mtb’s binding cleft and the specific chemical groups of the ligand suggests that KstR2Mtb is highly specific toward HIP-CoA. This is in line with the biophysical characterization of KstR2Mtb interactions with this ligand presented above. 73   Figure 3.2 Crystal structure of KstR2Mtb·HIP-CoA complex. (A) Overall structure. Chain A from the crystal’s AU is colored dark blue and chain B, produced by crystallographic symmetry, is colored light blue. HIP-CoA is shown in pink sticks. (B). Details of interactions between KstR2Mtb and HIP-CoA. The shown electron density is the Fo-Fc simulated annealing map calculated in the absence of HIP-CoA. Dashes indicate hydrogen bonds; black circle, the non-crystallographic symmetry axis; and dashed arrow, the closest approach between the dimer-bound ligands.  3.3.3 Binding of HIP-CoA alters the conformation of KstR2 Effector-binding typically triggers conformational changes in TFRs. To evaluate whether HIP-CoA binding triggers similar changes in KstR2Mtb, structural characterization of the ligand-free form of the regulator is required. Attempts to obtain crystals of KstR2Mtb in the absence of HIP-CoA were unsuccessful. However, a structure of the ligand-free form of KstR2RHA1 from R. jostii RHA1 is available (PDB 2IBD). KstR2RHA1 shares 59% amino acid sequence identity with 74  KstR2Mtb including 19 of the 23 residues that interact with HIP-CoA. A superposition of the KstR2Mtb·HIP-CoA complex and KstR2RHA1 structures yielded an rmsd of 1.5 Å over 186 matching Cα atoms of their protomers (Figure 3.3A). Nevertheless, the ligand-binding clefts of these proteins were structurally different (Figure 3.3B), particularly surrounding the HIP moiety, suggesting that ligand binding induces the conformational differences observed in the KstR2Mtb·HIP-CoA complex. The most striking alterations involve the positions of Trp-166 and Tyr-108, which shift up to 4.7 Å to stack on either face of bicycloalkanone rings of HIP in KstR2Mtb·HIP-CoA (Trp-170 and Tyr-112, respectively, in KstR2RHA1) (Figure 3.3B). The conformational differences include shifts in the positions of helices α4, α6 and α7 that surround the HIP-binding pocket and translate to regions beyond. Helix α4 connects the N- and C- terminal domains in both KstR2 structures and helix α6 forms multiple contacts with α1 of the N-terminal DBD (Figure 3.2 and 3.3B). Accordingly, the shift in position of these helices leads to a 15° outward rotation of the DBD in the ligand-bound KstR2Mtb in comparison to this domain’s position in the ligand-free KstR2RHA1 structure. In turn, this results in a significant difference in the relative position of the DBDs in the context of the KstR2 dimer (Figure 3.3A). These differences are consistent with HIP-CoA binding inducing a conformational change in KstR2. To evaluate whether the conformational differences between the ligand-bound and ligand-free KstR2 structures would affect the interaction of the regulator with its operator DNA, the KstR2Mtb·HIP-CoA structure was compared with that of a TFR bound to its operator DNA. The closest suitable match retrieved by our PDB search was SlmA from Vibrio cholera (PDB: 4GCT [205]). SlmA and KstR2Mtb protomers super-imposed with rmsd 2.8 Å over 179 matching Cα atoms (Figure 3.4A) facilitating the identification of potential DNA-binding secondary structure 75  elements in KstR2Mtb. As is characteristic of TFRs, the SlmA dimer formed symmetric contacts in adjacent major grooves of its operator DNA through helices α2 (the recognition helix) and α3 in each protomer (Figure 3.4). The equivalent region in the KstR2Mtb structure (between residues 33 and 50) featured prominently exposed residues that could form contacts with DNA (i.e., Val-33, Ser-44 and Tyr-48). Importantly, the relative position of the recognition helices differed dramatically in the operator-bound SlmA and ligand-bound KstR2Mtb dimers (Figure 3.4B): in KstR2Mtb·HIP-CoA, this helix is positioned further away from the DNA major groove. Overall, these analyses suggest that the conformation of the DBD domain in KstR2Mtb·HIP-CoA is not compatible with binding to its operator. This further suggests that HIP-CoA regulates the DNA-binding activity of KstR2 in the same manner as that established for other TFRs where effector-binding induces conformational changes that result in relieving the binding of the TFR to its operator DNA. 76   Figure 3.3 Conformational differences between KstR2Mtb·HIP-CoA and ligand-free KstR2RHA1 KstR2Mtb·HIP-CoA and ligand-free KstR2RHA1 are blue and brown, respectively. Chains A and B are dark and light shades, respectively. HIP-CoA is shown as pink sticks. (A). Overall comparison of the two conformations. Helices α2 and α3 are the putative DNA binding elements (helix-turn-helix motif). Inset shows a detail of the helix-turn-helix motif and the positional difference of a representative residue (KstR2Mtb Leu-43’ / KstR2RHA1 Leu-47’). (B). Localized conformational changes upon HIP-CoA binding. Single chains of the KstR2 dimer are shown for clarity. The positions of helices α1, α4, α6 and α8 differ between ligand-bound and ligand-free KstR2 structures 77   Figure 3.4 . Comparison of KstR2 structures with a TFR·DNA complex Superposition of KstR2Mtb·HIP-CoA (blue), KstR2RHA1 (red) and SlmA (green, PDB 4GCT) bound to its operator DNA. Superposition performed on subunit chains colored white, which adopt the same conformation. The recognition helices (α2), which adopt different positions, are shown as cylinders.  3.3.4 Functional validation of KstR2:HIP-CoA interactions To functionally validate the KstR2Mtb·HIP-CoA structural model, two key HIP-CoA binding residues were individually substituted using directed mutagenesis and the resulting KstR2Mtb variants were characterized using ITC and EMSA. More specifically, the structural data indicate that Arg-162 and Trp-166 form important interactions with HIP-CoA (Figure 3.2B) and that their substitution with methionine and leucine, respectively, should disrupt the binding of KstR2Mtb to its effector but not to its operator DNA. 78  Isotherms showed that both KstR2 variants were significantly impaired with respect to HIP-CoA binding. The R162M variant bound HIP-CoA with an affinity ~200 times lower than WT (Table 3.1). Like WT, HIP-CoA binding to the R162M variant was enthalpically driven with an unfavorable entropic contribution (Table 3.1). Unlike WT, the isotherm of the R162M variant showed a shallow titration curve consistent with the variant not being saturated at a 3-fold molar excess of HIP-CoA (Figure 3.5A). The one-site equation fit poorly to the isotherm, yielding a stoichiometry of N=1.77. The W166L variant showed no titration with HIP-CoA: the generated heats were equal to background (Figure 3.5B).   Figure 3.5 Isotherms of KstR2Mtb variants. (A) and (B). show representative titrations of 400 μM R162M and W166L, respectively, with aliquots of 40 μM HIP-CoA (25 mM HEPES, 50 mM KCl, pH 7.5 at 25oC). The bottom panel shows the corresponding isotherms with the best fit of the single binding site model.   Using EMSA, both the R162M and W166L variants bound to the KstR2 operator sequence with comparable affinity as WT KstR2Mtb (Figure 3.6). More specifically, WT and variants formed DNA: protein complexes at a mole ratio of 1:1 and no protein-free DNA probe was detected at a ratio of 1:50 DNA:protein. Consistent with previous results, the binding of DNA by WT KstR2Mtb was relieved in the presence of 50 μM HIP-CoA. Consistent with the ITC results, 79  500 μM HIP-CoA was required to detectably relieve binding to DNA by R162M. Moreover, the W166L·DNA complex was not detectably disrupted even at high concentrations of HIP-CoA.   Figure 3.6 EMSA of KstRMtb and variants. Each lane contains 2 nM DIG-labelled DNA probe and the indicated amount KstR2Mtb and HIP-CoA. Additional experimental details are in Materials and Methods.  3.3.5 A KstR2 operator sequence binds two KstR2 dimers The oligomeric state of KstR2Mtb was investigated using SEC-MALS. KstR2Mtb eluted as a single peak (tR = 10.2 min; 25 mM HEPES, 50 mM KCl, pH 7.5) with a molecular weight of 42.9 ± 0.2 kDa determined using the Rayleigh ratio (Figure 3.7). This is within 10% of the predicted mass of the KstR2Mtb dimer (46 kDa). The small discrepancy between the two may be partly due to the elongated structure of KstR2Mtb: the ASTRA6 software calculates molecular weight from molecular radius using spherical structures. To investigate the oligomeric state of KstR2Mtb bound to its operator, a DNA fragment containing a 14-bp KstR2 box flanked by 5 bp 80  on either side was synthesized. A sample of KstR2Mtb incubated with this 24-bp fragment yielded a single protein-containing peak (tR = 8.5 min) with a molecular weight of 97 ± 1 kDa. This is within 10% of the molecular weight predicted for a complex of two KstR2Mtb dimers and one DNA fragment (106 kDa). The DNA fragment alone (14.7 kDa) eluted at 10 min and caused negligible light scattering (data not shown).    Figure 3.7 SEC-MALS of KstR2Mtb Rayleigh ratios of 80 μM KstR2Mtb with (dotted line) or without (solid line) 20 μM DNA (a 24-bp duplex containing a KstR2 box) resolved using a Superdex 200 5/150 column. Calculated molecular weights from the corresponding peaks are shown on the right axis. Results are representative of two independent experiments 81   Elucidation of HIP catabolism in M. tuberculosis and actinobacteria Chapter 4:4.1 Introduction The pathway(s) by which steroid Rings C and D are degraded in bacteria is largely unknown. Work by Casabon et al. (2013) and that presented in Chapter 3, strongly implicate the ~14 genes of the KstR2 regulon, in the degradation of steroid Rings C and D in Actinobacteria [83, 91, 177]. Rings C and D degradation starts with the CoA thioesterification of HIP to HIP-CoA by FadD3 [79]. Given the absence of a CoA hydrolase in the cluster and the large number of β-oxidative enzymes predicted to be encoded by the KstR2 regulon, subsequent catabolic steps are predicted to involve CoA thioesterified catabolites.  Herein, the catabolism of HIP was elucidated. Using a number of mutants in Mtb, M. smegmatis, and RHA1 unique HIP catabolites were identified by developing a LC-MS based approach to analyze intracellular CoA thioesters. Novel catabolites were confirmed using a combination of NMR and chemical synthesis. Many of the HIP catabolic steps were reconstituted in vitro using recombinantly expressed enzymes encoded within the KstR2 regulon. The presented data provide the first description of the bacterial catabolism of steroid Rings C and D.  The work outlined in Chapter 4 was published in mBio in 2017 [178]. This study was highly collaborative. A complete list of author contributions is available in the preface. In brief, much of the LC-MS data was generated by Dr. Israel Casabon with help from Jason Rogalski from the Foster Lab at UBC. Experiments relating to the growth of Mtb were performed by Kirstin Brown or Jie Liu. Chemical synthesis was performed by Timothy Hurst from the Snieckus Lab at Queen’s University, Ontario.  82  4.2 Materials and methods 4.2.1 Chemicals and reagents ATP, p-coumaric acid, propionic anhydride, succinic anhydride, CoASH, phorbol 12-myristate 13-acetate (PMA), sodium acetate, sodium propionate, and cholesterol (>99%) were purchased from Sigma-Aldrich. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. ACCUZYME was purchased from Bioline. All other reagents were of HPLC or analytical grade. Buffers and solvents were prepared as previously described [79]. 4.2.1.1 Preparation of steroid metabolites and CoA thioesters HIP and HIP-CoA were prepared as described previously [79, 91]. p-Coumaroyl-CoA was synthesized as described using CouL [62] and was quantified spectrophotometrically in 50 mM sodium phosphate, pH 7.1 (333 = 21,000 M-1 cm-1) [206]. Propionyl-CoA and succinyl-CoA were synthesized from their corresponding anhydride as previously described [207]. 5α-OH HIC-CoA was synthesized from 5α-OH HIC using the mixed anhydride method [48]. Total synthesis of the 5-OH HICs will be published elsewhere. Briefly, 5 mg 5αOH-HIC dissolved in 0.5 ml dry THF was reacted with 10 μl ethylchloroformate and 26 μl triethylamine (TEA) for 20 min at room temperature. The resulting mixed anhydride was filtered through glass wool into 15 mg of CoASH dissolved in 1 ml THF:water (2:3, v:v), adjusted with NaOH to ~pH 8, and reacted at 37oC for 1 h. The reaction was stopped with the addition of 20 μl acetic acid and THF was removed under nitrogen. Typical mole yields were 80%. CoA thioesters were HPLC-purified using a Luna 3 µm PFP(2) 50 × 4.6 mm column (Phenomenex) equilibrated with 0.1 M ammonium acetate, pH 4.5. CoA thioesters were eluted using a 20 min linear gradient of 0 to 90% methanol in 0.1 M ammonium acetate, pH 4.5. The 83  eluate was monitored at 260 nm. Methanol was removed under N2 and compounds stored at -80°C. CoASH, acetyl-CoA, propionyl-CoA succinyl-CoA, 5α-OH HIC-CoA, and HIP-CoA were quantified at 260 nm using an 260 of 11,900 M-1 cm-1 in 50 mM sodium phosphate, pH 7.1. The identities of the CoA thioesters were verified by LC/MS/MS using a Zorbax SB300-C18 150 × 0.075 mm column (Agilent Technologies) and an Agilent 6550 ToF mass spectrometer operated as described in Section 4.2.6.2. 4.2.1.1.1 Purification of COCHEA-CoA COCHEA-CoA was obtained from ΔipdAB RHA1 using a protocol similar to that described above in the preparation of steroid metabolites to prepare CoA thioester metabolites for MS analysis with the following modifications. Phospholipid-free CoA metabolomes prepared from 10 × 4 l of ΔipdAB RHA1 were pooled, dried using a SpeedVacTM, suspended in 1 ml water and filtered using 0.2 μm PTFE membrane. COCHEA-CoA was purified from the CoA metabolome using a HP1100 series HPLC (Agilent Technologies) equipped with a Luna 3u PFP(2) 50 x 4.6 mm column (Phenomenex) operated at 1 ml min-1 and separated over a gradient of 0-60% methanol (90%) in 100 mM ammonium acetate, pH 4.5 over 12 min. COCHEA-CoA eluted at 8.4 min as a single species with a λmax = 250 nm. HPLC-purified COCHEA-CoA fractions were dried using a SpeedVacTM, suspended in 2 ml water and dialyzed against 2 l of water using a 100-500 Da cellulose ester dialysis membrane (Spectrum Laboratories Inc.). Desalted COCHEA-CoA was dried using a SpeedVacTM, washed and dried twice in deuterated methanol, then dissolved in 450 μl deuterated water (D2O). The final concentration of COCHEA-CoA was estimated to be 600 μM as determined using an HPLC standard curve of CoASH (ε260 = 11.9 mM-1 cm-1) and using an extinction coefficient of 16.4 mM-1 cm-1 due to the additional 84  absorbance from a double bond [86]. Each biotransformation, using cells from 4 l of culture, yielded ~48 nmol of COCHEA-CoA. COCHEA-CoA was confirmed via LC/MS/MS prior to NMR. 4.2.1.1.2 Purification of MOODA MOODA was purified from the supernatant of cholesterol-incubated ΔfadE32 M. smegmatis as follows. Four × 1 l cultures were grown to mid log (OD600 = 0.6) in 7H9 media + 0.5% Tween 20 + 0.2% glycerol and harvested by centrifugation (4000 × g, 20 min at 16oC). Cells were washed once using M9 salts, suspended in 200 ml M9 salts, 2 mM MgSO4, 0.1 mM CaCl2 and 0.5 mM cholesterol, split into 2 × 100 ml, then incubated at 37oC for 24 h in 250 ml baffled flasks. Cells were harvested by centrifugation and discarded. The supernatant was collected, acidified to ~pH 2 using HCl and extracted 3× with 1:1(v:v) ethyl acetate. The organic phases were pooled, dried over anhydrous MgSO4 and filtered through Whatman paper.  Ethyl acetate was removed using a rotavap. The oily residue was dissolved in water and brought to pH 7 with NaOH. MOODA was purified using a Strata-X-A strong anionic exchange solid phase extraction column (Phenomenex) according to the manufacturer’s protocol. GC/MS analysis indicated that MOODA was >95% homogeneous. The yield of MOODA was ~125 μg l-1. For NMR characterization, ~0.5 mg MOODA was dried using a SpeedVacTM, washed twice with deuterated methanol (MeOD), and dissolved in 500 μl D2O. 4.2.2 DNA manipulation, plasmid construction, and gene deletions DNA was propagated, amplified, digested, ligated, and transformed using standard protocols [180]. Genes were amplified using the primers and template genomic DNA listed in Appendix A. Amplicons were digested with the enzymes indicated in the descriptions of the 85  oligonucleotides. The nucleotide sequence of all constructs was verified prior to their use. Plasmids based on pTipQc2 and pMV361.apr were electroporated into RHA1 and M. smegmatis, respectively, as previously described [79, 208].  Mycobacterial genes were deleted using homologous recombination [209]. Allelic exchange substrate (AES) constructs were generated using the oligonucleotides listed in Appendix A to amplify the up- and downstream regions of the genes to be deleted and cloning them on either side of the hygR cassette in pYUB854. The linearized AES was electroporated into Mtb and M. smegmatis harboring pJV53. In RHA1, mutants were obtained using a SacB-based selection as previously described [210]. Briefly, pK18-derived plasmids were electroporated into E. coli S17.1 and then conjugated into RHA1. After the second recombination, kanamycin-sensitive/sucrose-resistant colonies were screened and confirmed using PCR. 4.2.3 Growth of bacteria Strains and plasmids used in this study are provided in Appendix A. RHA1 strains were cultivated aerobically at 30°C on M9 mineral medium as described previously [79] containing either 1 mM cholesterol, 1.5 mM HIP, or 10 mM pyruvate. M. smegmatis strains were cultivated aerobically at 37°C in M9 supplemented with 2 mM MgSO4 and 0.1 mM CaCl2 as described previously [211]. Mtb strains were grown on 7H9 media supplemented with 0.5% tyloxapol and either 0.2% glycerol or 0.5 mM cholesterol as described previously [40]. Escherichia coli strains were grown at 37°C in Luria-Bertani (LB) broth. Hygromycin (150 µg ml-1 for E. coli, 50 µg ml-1 for mycobacteria), kanamycin (25 µg ml-1 for E. coli, 20 µg ml-1 for mycobacteria), ampicillin (100 µg ml-1), chloramphenicol (34 µg ml-1) and apramycin (30 µg ml-1) were used for selection 86  where appropriate. Growth of RHA1 and M. smegmatis was followed using OD600 nm. Growth of Mtb was followed using OD600 nm and CFU ml-1 by serially diluting in saline containing 0.05% tween-80 and plating on 7H10 + OADC plates. 4.2.4 Macrophage infections THP-1 cells (American Type Culture Collection, TIB-202), were cultured in GIBCO® RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1 mM sodium pyruvate and were maintained between 2 and 5 x 105 cells ml-1. Mtb strains were grown to late log phase in Middlebrook 7H9 supplemented with OADC, aliquots were frozen at -80°C, and CFU ml-1 was enumerated. THP-1 cells were seeded in 24-well flat-bottom tissue culture plates and allowed to adhere in the presence of 50 ng ml-1 PMA for 48 hours at 37°C in a humidified, 5% CO2 atmosphere. Cells were washed to remove PMA and incubated for a further 48 hours prior to infection. Bacteria were added to THP-1 cells at an MOI of 1:1 for 6 hours. Cells were washed three times to remove extracellular bacteria and were then incubated for 7 days. At each time point, THP-1 cells were lysed by adding 0.06% SDS. Bacteria were then serially diluted in saline containing 0.05% tween-80, and plated on Middlebrook 7H10 with OADC for enumeration. 4.2.5 Preparation of CoA metabolomes Cells were grown in 900 ml pyruvate or glycerol minimal medium as described above. Cells were harvested at mid-log phase, washed with fresh medium lacking growth substrate, then suspended in 100 ml growth medium supplemented with either 0.5 mM cholesterol, 20 mM pyruvate (RHA1) or 0.2% glycerol (M. smegmatis and Mtb). Biotransformations were incubated for 48 hours at 30°C/200 rpm for RHA1 and 37°C/200 rpm for M. smegmatis and 37°C in roller 87  bottles for Mtb. Cells were cooled on ice, harvested by centrifugation, washed once with ice-cold minimal medium, and then stored at -80°C until use.  CoA thioesters were extracted from cell pellets using a modified protocol described for eukaryotic cells [212]. Preparations were kept on ice or 4°C unless otherwise noted. Frozen cell pellets were suspended in 4 ml of acetonitrile:isopropanol (3:1 v:v) containing 15-50 nmol p-coumaroyl-CoA. Cells were disrupted using a FastPrep-24 bead beater (6 × 40 s at 6.5; 5 min pauses on ice between rounds). After the first three rounds, 0.1 M KH2PO4, pH 6.7 was added to a final concentration of ~25 mM KH2PO4. The supernatant was recovered by centrifugation (15,000 × g for 30 min), filtered through a 0.2 µm regenerated cellulose membrane (Phenomenex), and acidified with 0.25 ml glacial acetic acid per ml of extract. The acidified extract was applied to a 100 mg 2-(2-pyridyl)ethyl-functionalized silica column (Supelco, 54127-U), equilibrated using 1 ml “equilibration” solution (acetonitrile:isopropanol:water:acetic acid (9:3:4:4 v:v:v:v)) at -20°C. The resin was washed with 2 ml equilibration solution at -20°C before CoA metabolites were eluted with 2 ml methanol:0.25 M ammonium acetate, pH 7 (4:1 v:v) at -20°C. Methanol was evaporated under N2, then the sample was flash frozen in liquid N2 and lyophilized overnight. The lyophilized sample was suspended in 0.6 ml methanol, deposited on a Phree column (Phenomenex) to remove phospholipids and recovered by centrifugation (500 × g for 10 min). This sample eluate was dried under N2, suspended in 0.2 ml methanol, and stored at -80°C. Immediately prior to LC/MS analysis (see below), the cellular extracts was diluted 10-fold in 0.1 M ammonium acetate, pH 4.5 and filtered 0.2 µm (PTFE). 4.2.6 CoA metabolite profiling by LC-MS 4.2.6.1 Targeted LC-MS CoA thioesters were detected in cellular extracts using an Agilent 6460 Triple Quadrupole (QQQ) operated in positive ion mode and connected to a 80 x 0.25 mm Luna 3 μm PFP(2) (Phenomenex) analytical column through a 15 x 0.25 mm PFP(2) trapping column. CoA 88  thioesters were separated using a gradient of 100 mM ammonium acetate, pH 4.5 into 20 mM ammonium acetate, pH 4.5 in 98% methanol over 30 min, operated at 3 μl min-1. A mixture of CoA thioester standards was run prior to each CoA metabolome to verify column performance and MRM sensitivity. Collision Energy Dissociation (CID) and fragmentor voltages were selected based on signal optimization using CoA thioester standards.  High resolution MS, MS2 and MS3 analyses of CoA thioesters were performed in positive ion mode on a Bruker Impact-II Q-ToF equipped with a 150 x 0.25 mm Luna 3 μm PFP(2) (Phenomenex) column. CoA thioesters were eluted using a gradient of 100 mM ammonium acetate in 2% methanol and 20 mM ammonium acetate in 98% methanol. The mass spectrometer was calibrated daily. 4.2.6.2 Untargeted LC-MS To ensure that no CoA thioesters were missed using our targeted analysis method, representative CoA metabolomes were analyzed using LC/MS/MS as previously described [150]. Briefly, cellular extracts were diluted 1:49 with acetonitrile:water (3:97 v:v) supplemented with 0.1% formic acid then injected onto a Zorbax SB300-C18 150 x 0.075 mm column (Agilent Technologies) operated at 0.3 μl min-1 and eluted using a 10 min gradient from 3 - 97% acetonitrile. Mass spectra were recorded in positive ion mode on an Agilent 6550 Time of Flight (ToF) mass spectrophotometer using a scanned mass range of 50-1100 Da. Species were determined to be CoA thioesters based on the characteristic [M+H]+-507 and 428 m/z fragments. 4.2.6.3 NMR characterization of metabolites 1H-NMR, 1H-13C HMBC, 1H-13C HSQC, 1H-1H COSY, and 1H-1H TOCSY spectra were recorded at 25oC using a Bruker 850 MHz NMR spectrometer. 1H-NMR spectra were recorded 89  before and after each experiment to ensure no degradation had occurred during data collection. NMR data were analyzed using the ACD/NMR Processor v12.0 Academic Edition software. 4.2.6.4 Analysis of CoA metabolomics data Peak integration, retention time, and signal-to-noise ratio (S/N) were calculated using MassHunter Qualitative Analysis B.06.00 (Agilent Technologies). Peaks were defined as having S/N > 3. Analysis of CoA metabolomic data was completed using the [M+H]  -507 m/z transitions due to the higher signal intensity as compared to the [M+H]  428 m/z transitions, although the latter transition was confirmed for each CoA thioester characterized. CoA thioester levels were normalized to the internal standard (p-coumaroyl-CoA) prior to calculating their relative concentrations and proportion of the total cellular CoA pool. 4.2.7 Protein production and purification Proteins were produced in and purified from E. coli Rosetta 2 pLysS (IpdFMtb), E.coli BL21 (DE3) (MBP-IpdCDOC21) or RHA1 (EchA20RHA1, FadA6Mtb and IpdABRHA1). Cells were grown on LB supplemented with carbenicillin, ampicillin or chloramphenicol at 50, 100 and 34 μg ml-1, respectively, as appropriate. Single colonies of freshly transformed cells were used to inoculate 50 ml growth medium and incubated overnight at 37oC, 200 rpm (E. coli) or 30oC, 200 rpm (RHA1). Ten ml of overnight culture was used to inoculate 1 l fresh medium. At an OD600 of ~0.6, inducer was added (0.5 mM IPTG for E. coli; 10 μg ml-1 thiostrepton for RHA1) and cultures were incubated for an additional 16 h, then cells were harvested by centrifugation. For E. coli cultures, this 16 h incubation was done at 25oC. In addition, E. coli cultures producing MBP-IpdCDOC21 were supplemented with 0.1% glucose to repress intracellular expression of amylases. Cell pellets were stored at -80oC until use. 90  To purify the various proteins, cell lysis buffers contained 2 U ml-1 DNaseI and one tablet protease inhibitor cocktail (Roche). E. coli cells were lysed using five passages through an Avestin Emulsiflex-05 homogenizer operated at 10,000 p.s.i. and RHA1 cells were lysed using an MP Biomedicals FastPrep-24 bead beater (five rounds of 40 s). Cell lysates were clarified by ultracentrifugation (40000 × g, 45 min at 4oC) then filtered through a 0.45 μm membrane. Proteins were buffer-exchanged and concentrated using a Centricon 30 K (Millipore) or an Amicon Stirred Cell (Millipore) equipped with a 30 K regenerated cellulose Ultrafiltration Membrane (Millipore), then flash frozen in liquid nitrogen. Protein preparations were evaluated using SDS PAGE.  To purify IpdFMtb, pellets of E.coli Rosetta 2:pETRv3559c were suspended in 25 ml 50 mM sodium phosphate, pH 8.0, 10% glycerol. The clarified lysate was loaded onto 3 ml Ni-Sepharose 6 Fast Flow resin (GE Healthcare) and IpdFMtb was eluted using a gradient of 10-500 mM imidazole in 50 mM sodium phosphate, pH 8.0 according to the manufacturer’s protocol. Fractions containing IpdFMtb were pooled, dialyzed overnight against 25 mM HEPES, pH 7.5, 50 mM KCl and 10% glycerol, and concentrated to ~15 mg ml-1. To purify MBP-IpdCDOC21, pellets of E.coli Rosetta2: pMALDOC21 were suspended in 30 ml 50 mM Tris, pH 8.0, 100 mM NaCl. The clarified lysate was loaded onto a 15 ml column of amylose resin (New England Biolabs). The resin was washed with ~75 ml 50 mM Tris, pH 8.0, 100 mM NaCl (Buffer A), then MBP-IpdCDOC21 was eluted using ~75 ml Buffer A containing 20 mM maltose. Fractions containing >70% MBP-IpdCDOC21 were pooled, concentrated to ~3 ml and exchanged into Buffer A. MBP-IpdCDOC21 was precipitated using a final concentration of 1.75 M ammonium sulfate in Buffer A. The white precipitate was collected 91  via centrifugation (4000 × g, 5 min, 4oC), washed with fresh Buffer A containing 1.75 M ammonium sulfate and solubilized in 1 ml Buffer A.  To purify EchA20RHA1, FadA6Mtb, and IpdABRHA1, pellets RHA1: pTipR1EchA20, RHA1:pTipFadA6, RHA1:pTipR1IpdAB, respectively, were suspended in 25 ml 50 mM sodium phosphate, pH 8.0 containing 300 mM NaCl and 10 mM imidazole. Clarified lysates were loaded onto 2 ml Ni-Sepharose G Fast Flow resin (GE Healthcare) and eluted using a gradient of 10-500 mM imidazole according to the manufacturer`s protocol. Fractions containing the desired protein were pooled and dialyzed overnight against either 25 mM HEPES, pH 7.5 and 50 mM NaCl (FadA6Mtb and IpdABRHA1) or 25 mM HEPES, pH 7.5, 300 mM NaCl, 1 mM MgCl2, 1 mM NaHCO3 and 10% glycerol (EchA20RHA1). Proteins were concentrated to ~10 mg ml-1. 4.2.8 Enzymatic transformations Assays using purified enzymes were performed in a final volume of 100 μl containing 10 mM sodium phosphate, pH 8.0, 100 μM 5α-OH HIC-CoA, 100 μM NAD+, 5 μM FMN and 2 μM of each relevant enzyme. Assays containing FadA6Mtb also contained 50 μM CoASH. Reactions were incubated at 37oC for 1 h. Proteins were removed before HPLC and MS analysis by the addition of 200 μl acetonitrile + 5% acetic acid. Volatile solvents were removed using a SpeedVacTM concentrator and precipitated proteins were pelleted by centrifugation (16,000 × g, 5 min, 4oC). Samples were filtered through a 0.2 μm membrane, diluted 2:1 in water and run on an HP1100 series HPLC equipped with a Luna 3u PFP(2) column. The eluate was monitored at 260 nm. The identity of new peaks was confirmed by LC/MS/MS as described above.  92  4.2.9 Bioinformatic analyses The amino acid sequences of Rings C/D catabolic enzymes were obtained from the NCBI using their Mtb H37Rv gene loci. These sequences were used to search for homologs in the genomes of RHA1, M. smegmatis MC2155, C. testosteroni CNB-2, and S. denitrificans DSM 18526 using BLAST-P. Best hits were used to search the Mtb H37Rv genome to evaluate whether they were reciprocal best hits. Closest characterized homologs were determined by using the amino acid sequences of Mtb Rings C/D catabolic enzymes to search against the Protein Data Bank database using BLAST-P and manually identifying the best characterized result for each enzyme. 4.3 Results 4.3.1 The ipdABC genes are required for growth on cholesterol and HIP The KstR2 regulon has been strongly implicated in the catabolism of HIP in mycolic acid-containing Actinobacteria [91]. Because FadD3, encoded by the KstR2 regulon, initiates HIP catabolism, we hypothesized that the other regulon-encoded enzymes act downstream of FadD3. Deletion mutants of ipdAB and ipdC in each of Mtb (Rv3551-Rv3553) and RHA1 (RHA1_RS22695-22685) were initially focused on to gain insights into HIP catabolism.   Figure 4.1 Growth of ΔipdAB Mtb. 93  WT Mtb Erdman (black), ΔipdAB Mtb (red), or ΔipdAB Mtb::ipdAB (blue) were grown on (A) 0.5 mM cholesterol, (B) 0.2% glycerol, or (C) in PMA-differentiated THP-1 cells. Data represent the mean of biological triplicates  An ipdAB mutant constructed in Mtb Erdman did not grow on cholesterol (Figure 4.1A), but grew as the wild-type on glycerol (Figure 4.1B). The growth defect on cholesterol was restored through complementation. Because mutations in Mtb can exhibit unexplained strain differences [213] an equivalent ipdAB mutant constructed in Mtb CDC1551 was verified to have the same phenotype as ipdAB Mtb Erdman (data not shown). Finally, an ipdAB mutant of RHA1 exhibited a similar phenotype: it did not grow on either HIP or cholesterol, and grew normally on pyruvate (Figure 4.2). The growth defects on cholesterol and HIP were restored through complementation with ipdAB of Mtb.  94   Figure 4.2 Growth and CoA metabolites of RHA1 strains Growth of WT::pTip-Qc2 (blue), ΔipdAB ::pTip-Qc2 (red), ΔipdAB:: pTipCoL51 (red, dashed), ΔipdC::pTipQc2 (green) and ΔipdC:: pTipRv3553 (green, dashed) on: (A) 10 mM pyruvate; (B) 1 mM cholesterol; (C) 1.5 mM HIP; and (D) 1 mM HIP plus 10 mM pyruvate. (E) Depletion of HIP by RHA1 strains, color-coded as in growth curves as measured by GC/MS and reported as % of initial levels. Data are the mean of triplicates. Error bars show standard deviation. Data for panel D were acquired using a BioScreen C (Growth Curves USA)  An ipdC mutant of RHA1 also did not grow on either cholesterol or HIP but grew normally on pyruvate (Figure 4.2). The growth defect on cholesterol and HIP was restored through complementation with ipdCMtb. Gas chromatography-coupled mass spectrometry (GC/MS) established that while cholesterol and HIP were depleted in the wild-type and complemented strains, they were not detectably depleted by the ipdC mutant (Figure 4.2E). Similar results were obtained in Mtb Erdman: the ipdC mutant did not grow on cholesterol but 95  grew normally on glycerol (Figure 4.3). Moreover, this phenotype was restored through complementation with an integrative plasmid harboring ipdC.  Figure 4.3 Growth and CoA metabolites of ΔipdC Mtb. WT (black), ΔipdC (red), ΔipdC::ipdC (blue) Mtb Erdman were grown on (A) 1 mM cholesterol; (B) 0.2% glycerol; or (C) 0.5 mM cholesterol and 0.2% glycerol. (D) CoA metabolome of ΔipdC (red) and WT Mtb (blue) incubated with 0.5 mM cholesterol. Arrows indicate the peaks corresponding to the 5-OH HIC-CoA in the ΔipdC RHA1 CoA metabolome.  4.3.2 Growth in macrophages Transposon mutagenesis studies suggest that ipdA is essential for Mtb survival in macrophages [52]. Moreover, the gene is essential for survival of R. equi in foals [23]. Therefore the growth of ipdAB Mtb in PMA-differentiated THP-1 cells was tested (Figure 4.1C). WT Mtb increased >350-fold over 7 days, corresponding to a doubling time of 19.6 hours. The mutant increased ~10-fold over this time, corresponding to a doubling time of 46.5 hours, while complementation restored intracellular replication to 131-fold. These results are consistent with Mtb catabolizing cholesterol during intracellular growth [134]. 4.3.3 The accumulation of cholesterol catabolites in the ipd mutants In an attempt to identify the respective substrates of IpdAB and IpdC, the occurrence of metabolites in the RHA1 mutants was investigated. GC/MS analyses revealed that, when incubated with cholesterol, ipdC RHA1 accumulated low amounts of a metabolite with an m/z 96  of 356 (Figure 4.4A). No metabolites were detected in the culture supernatant when cells of ipdAB RHA1 were incubated with cholesterol (Figure 4.4A). Failure to detect significant amounts of extracellular metabolites in the supernatants of cholesterol-incubated ipdC and ipdAB mutants was hypothesized to be due to the accumulation of intracellular, CoA thioesterified metabolites that are not readily excreted. To test this hypothesis, CoA thioesters were extracted from cells and analyzed them using LC/MS. LC was performed using a pentafluorophenyl (PFP) resin to maximize resolution of the CoA thioesters. Mtb, RHA1 and M. smegmatis cells incubated with various substrates contained CoASH, acetyl-CoA, propionyl-CoA, and/or succinyl-CoA irrespective of the growth substrate (Table 4.1). The identity of these metabolites was based on their m/z-values and their retention time (Rt) on the PFP column with respect to synthetic standards. Their concentrations were quantified relative to p-coumaroyl-CoA, the internal standard. Some strains also contained low amounts of dephospho-CoASH depending on the substrate, as has been reported in other cells [150]. When incubated in the presence of cholesterol, the ipd mutants accumulated CoA thioesters that were not detected either in the wild-type strains or in ipd mutants incubated with glycerol or pyruvate (data not shown). More specifically, cholesterol-incubated cells of ΔipdC Mtb and RHA1 contained significant amounts of two CoA thioesters with m/z values of 962, one of which was more abundant than the other (Figure 4.4B, Table 4.1). The main CoA thioester that accumulated in cholesterol-incubated cells of ΔipdAB Mtb and RHA1 eluted with a Rt of 22.7 min and had an m/z value of 976 (Figure 4.4C, Table 4.1).  97  Table 4.1 Characterization of CoA metabolites found in this study Compound Full Name M+H (QQQ)a MRM Transition M+H (Bruker)b Molecular Formula Retention time (min) NMR Confirmed Reference Major peak observed in CoA metabolomec in vitro enzymatic productiond CoASH Coenzyme-A 768.1 768=> 261,428 768.1210 C21H37N7O16P3S+ 17.0-18.4 Yes SIGMA All  Dephos-CoASH Dephospho-CoASH 688.1 688=>348 688.1562 C21H36N7O13P2S+ 19.3 No All Acetyl-CoA   810.1 810=> 303,428 810.1315 C23H39N7O17P3S+ 19.9-20.9 yes SIGMA All COCHEA-CoA + IpdABRHA1 + FadA6Mtb Propionyl-CoA 824.1 824=> 317,428 824.1466 C24H41N7O17P3S+ 21.6-22.9 Yes All Succinyl-CoA 868.1 868=> 361,428 868.1400 C25H41N7O19P3S+ 21.6-22.2 Yes All Unknown Unknown 992.2 992=>  485,428 992.1901 C32H49N7O21P3S+ 20.8-22.0 No  RHA1 ΔipdAB Mtb ΔipdAB Δ MC2155 ΔipdAB  5β-OH HIC-CoA 3aβ-H-4α(Carboxylic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone 962.1 962=> 455,428 962.2159 C32H51N7O19P3S+ 22.0-22.9 Yes (5βOH-HIC) This Study RHA1 ΔipdC, ΔipdABC  Mtb ΔipdC  HIEC-CoA (7aS)-7a-Methyl-1,5-dioxo-2,3,5,6,7,7a-hexahydro-1H-indene-4-carboxyl-CoA 958.1 958=> 451,428 958.1899 C32H47N7O19P3S+ 23.2-23.4 No This Study MC2155 ΔechA20 5α-OH HIC-CoA + IpdCDOC21+IpdFMtb 98   Compound Full Name M+H (QQQ)a MRM TransitionM+H (Bruker)b Molecular Formula Retention time (min) NMR Confirmed ReferenceMajor peak observed in CoA metabolomecin vitro enzymatic productiondCOCHEA-CoA (3R)-2-(2-Carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA 976.2 976=> 469,428 976.1920 C32H49N7O20P3S+ 22.7-23.4 Yes This Study RHA1 ΔipdAB MC2155 ΔipdAB Mtb ΔipdAB HIEC-CoA + EchA20RHA1MOODA-CoA 4-Methyl-5-oxooctanedioyl-CoA 952.2 952=> 445,428 952.1960 C30H49N7O20P3S+ 23.0-23.2 Yes (MOODA) This Study MC2155 ΔfadE32 (MOODA in Sup.)COCHEA-CoA + IpdABRHA1 + FadA6Mtb5α-OH HIC-CoA 3aα-H-4α(Carboxylic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone 962.1 962=> 455,428 962.2159 C32H51N7O19P3S+ 23.0-23.8 Yes (5αOH-HIC) This Study RHA1 ΔipdC, ΔipdABC  Mtb ΔipdC MC2155  ΔechA20, ΔipdF 3'oxo-5OH-HIP-CoA  1004.2 1004=> 453,428 1004.2265 C34H53N7O20P3S+ 23.3-24.1 No This Study   3',5-diOH-HIP-CoA  1006.2 1006=> 499,428 1006.2420 C34H55N7O20P3S+ 24.0-24.2 No This Study   99    Figure 4.4 Accumulation of cholesterol derived metabolites from ΔipdAB and ΔipdC strains. (A) GC/MS traces of culture supernatants of ΔipdC, ΔipdAB, ΔfadD3ipdAB and WT RHA1 incubated with cholesterol. Peaks 1 and 2 correspond to TMS-5α-OH HIC and TMS-HIP, respectively. CoA metabolome of cholesterol-incubated cells of (B) WT (blue) and ΔipdC (red) RHA1 or (C) WT (blue) and ΔipdAB (red) Mtb. The major unique peaks in the ΔipdC and ΔipdAB metabolomes correspond to 5αOH-HIC-CoA and COCHEA-CoA, respectively (inset). Lighter shaded curves in panels B and C are based on the 768=>261 transition observed in free CoASH as well as CoA thioesters subjected to in-source fragmentation. 100   4.3.4 Identification of CoA metabolites in the ipd mutants The 962 m/z metabolite that accumulated in ΔipdC RHA1 and Mtb was predicted to be produced by the β-oxidative cleavage of acetyl-CoA from HIP-CoA and reduction of the 5-oxo group to yield 3aα-H-4α(3’-carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA; Figure 4.4B). To identify the metabolites that accumulated in the ΔipdC mutants, the 5 and 5 isomers of 5-OH HIC were synthesized and confirmed by NMR (Appendix C). Each isomer was thioesterified to yield 5- and 5-OH HIC-CoA, respectively, which were then purified by HPLC. The high resolution [M+H]+ m/z value and MS3 fragmentation pattern (of the [M+H]+-507 fragment), of synthetic 5-OH HIC-CoA corresponded to those of the most abundant CoA thioesters in cholesterol-incubated ΔipdC mutants. The presence of both α and β diastereomers was confirmed by the Rt values of their corresponding standards. Interestingly, the m/z value of the metabolite that accumulated in the supernatant of cholesterol-incubated ΔipdC RHA1 corresponds to that of 5-OH HIC (Figure 4.4A).  The most abundant CoA thioester in cholesterol-incubated ΔipdAB mutants accumulated in sufficient quantities to allow its isolation from the RHA1 mutant for further characterization. The metabolite was identified as 2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) based on high resolution mass spectrometry (976.1960 m/z), 1H, COSY, TOCSY, HMBC, and HSQC NMR (Appendix C). Most diagnostically, the carbon-11 (C-11, see Figure 4.4C for numbering of carbons) methyl protons appear as a doublet, establishing that C-5 bears a hydrogen and that Ring D is open. C-10 is thioesterified based on C-1 having the same 13C NMR chemical shift as in 2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1 [214].  101  4.3.5 Enzymatic transformation of 5-OH-HIC-CoA To further elucidate the catabolism of HIP, various KstR2-encoded enzymes were purified, including IpdAB, IpdC, IpdF, EchA20 and FadA6. These preparations were used to evaluate their abilities to transform 5-OH HIC-CoA in vitro. I initially sought to work exclusively with the Mtb homologs. However, I was unable to obtain all of the Mtb homologs in stable, soluble forms despite testing various host strains and expression conditions. In some cases, the RHA1 homolog was more stable. For IpdC, I obtained the best preparations using the homolog from P. putida DOC21, a bile acid-degrading bacterium. The gene encoding IpdCDOC21, DC0014-19, is the reciprocal best hit of ipdCMtb and occurs in a predicted operon similar in structure to that of S. denitrificans which contains ipdAB. SDS-PAGE analyses of the various protein preparations are provided in Figure 4.5. The subscripts Mtb, RHA1 and DOC21 identify the parent strain of each enzyme. Transformation experiments were performed by incubating synthetic 5-OH HIC-CoA with various enzymes and characterizing the reaction products using LC/MS. 102   Figure 4.5 Electrophoretic analyses of gene deletion mutants and purified proteins. (A) PCR confirmation of ΔipdAB in RHA1 and Mtb; ΔipdC in RHA1 and Mtb; ΔipdF, ΔechA20, and ΔfadE32 in M. smegmatis using the listed primer sets (Appendix A). Thick bands in DNA ladder depict 3 kb (top) and 1 kb (bottom). Gene deletions in mycobacteria introduced a ~1.5 kb hygR marker whereas RHA1 gene deletions are markerless (Section 4.2.2).  (B) SDS PAGE loaded, from left to right, with 0.5 μg each of, MBP-IpdCDOC21, IpdFMtb, EchA20RHA1, IpdABRHA1, and FadA6Mtb. Purified proteins are flanked by molecular weight standards.  Incubation of 5α-OH HIC-CoA with IpdFMtb and IpdCDOC21 yielded a compound with m/z value of 958 (Figure 4.6A and B; red trace), consistent with the oxidation of the 5-OH group of HIC and the introduction of a double bond. Interestingly, the two enzymes did not detectably transform 5β-OH HIC-CoA (data not shown). Based on its mass and the structure of the downstream metabolite, COCHEA-CoA, the IpdC/IpdF transformation product was 103  provisionally identified as (7aS)-7a-methyl-1,5-dioxo-2,3,5,6,7,7a-hexahydro-1H-indene-4-carboxyl-CoA (HIEC-CoA). This assignment is consistent with the function of the next step of the pathway, as discussed below. However, NMR data are required for a definitive identification. Neither IpdFMtb nor IpdCDOC21 transformed 5-OH HIC-CoA in the absence of the other enzyme. Moreover, as described below, ΔipdF M. smegmatis accumulated the same major metabolite as the ΔipdC mutants, 5α-OH HIC-CoA. Therefore, I was unable to determine the order of reaction of IpdF and IpdC. However, incubation of 5-OH HIP-CoA with IpdFMtb yielded HIP-CoA, demonstrating that this enzyme catalyzes oxidation of the 5-OH.  Incubation of 5-OH HIC-CoA with IpdFMtb, IpdCDOC21 and EchA20RHA1 yielded a compound whose Rt and m/z values were identical to those of COCHEA-CoA (Figure 4.6C; green trace), the major metabolite that accumulated in the ΔipdAB mutants.  Finally, incubation of 5-OH HIC-CoA with IpdFMtb, IpdCDOC21, EchA20RHA1, IpdABRHA1 and FadA6Mtb yielded a compound with m/z value of 952 (Figure 4.6D; blue trace). Hydrolysis of the CoA thioester yielded a compound that GC/MS revealed to be 4-methyl-5-oxo-octanedioc acid (MOODA), which accumulated in a ΔfadE32 mutant of M. smegmatis when incubated with cholesterol (Figure 4.7). MOODA-CoA has a predicted m/z value of 952. Consistent with such a role, the enzymatic transformation of COCHEA-CoA to MOODA-CoA required CoASH and yielded stoichiometric amounts of acetyl-CoA (Figure 4.6). 104   Figure 4.6 LC/MS analyses of the transformation of 5-OH HIC-CoA by purified enzymes. Left panels show HPLC traces of reaction mixtures containing 100 μM 5-OH HIC-CoA, 125 μM NAD+, 50 μM CoASH, 5 μM FMN (10 mM phosphate, pH 7.5) and: (A) no enzyme (control); (B) IpdFMtb and IpdCDOC21; (C) IpdFMtb, IpdCDOC21, and EchA20RHA1; or (D) IpdFMtb, IpdCDOC21, EchA20RHA1, IpdABRHA1 and FadA6Mtb. LC/MS analyses of the reaction products identified the major HPLC Major peaks are color-coded with fragmentation patterns in the right-hand panels and correspond to: 5α-OH HIC-CoA (962 m/z), HIEC-CoA (958 m/z), COCHEA-CoA (976 m/z), and MOODA-CoA (952 m/z). Other LC peaks correspond to acetyl-CoA (810 m/z) and (F) FMN.  105  4.3.6 Bioinformatic analysis of HIP catabolic enzymes Table 4.2 Annotation of KstR2 regulon Genea H37Rvb RHA1c M. smegd CNB-2e S. denitf Annotation of gene product Best hitg Identityh  Rv3548c RS22710 5999 1286 00355 Short chain type dehydrogenase/ reductase P22414 42  Rv3549c RS22705 6000 1330 12450i Short chain type dehydrogenase/ reductase A6CQL2 34 echA20 Rv3550 RS27700 6001 1280 00335 HIEC-CoA hydrolase 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB), P76082 28 ipdA Rv3551 RS22695 6002 1276 00310 COCHEA-CoA hydrolase, α subunit glutaconate CoA transferase, α subunit, Q59111 26 ipdB Rv3552 RS22690 6003 1277 00315 COCHEA-CoA hydrolase, β subunit glutaconate CoA transferase, β subunit, Q59111 25 ipdC Rv3553 RS22685 6004 1279 00330 5-OH HIC-CoA reductase Enoyl-[ACP] reductase II (FabK), Q9FBC530 fadA6 Rv3556c RS22430 6008 1283 00350 β-keto CoA thiolase 3-oxo-acyl CoA thiolase, I6XHI4 38 kstR2 Rv3557c RS22425 6009 - - HIP-CoA repressorj - - ipdF Rv3559c RS22420 6011 1289 00370 5-oxo HIC-CoA oxidase levodione reductase, Q9LBG2 39 fadE30 Rv3560c RS22415 6012 1288 00365 acyl-CoA dehydrogenase I6YCA3 31 fadD3 Rv3561 RS22410 6013 1360 09100i HIP-CoA synthetasej - - fadE31 Rv3562 RS22400 6014 1281 00340 acyl-CoA dehydrogenase I6YCA3 29 fadE32 Rv3563 RS22395 6015 1282 00345 MOODA-CoA dehydrogenase I6YCA3 23 fadE33 Rv3564 RS22390 6016 1287 00360 acyl-CoA dehydrogenase I6YCA3 23 aName assigned based on current study. Identification numbers for the corresponding genes in bMtb H37Rv, cRHA1 and dM. smegmatis, Msmeg_ omitted from IDs for simplicity. eC.testosteroni CNB-2, CtCNB2_ omitted from IDs for simplicity, fSteroidobacter denitrificans DSM 18526, ACG33_ omitted from IDs for simplicity. gAccession number of functionally characterized best hit in NCBI database. hAmino acid sequence identity of the Mtb enzyme and its experimentally characterized best hit based on full sequence alignment. iNot clustered with other Rings C/D catabolic genes. jMtb enzyme characterized. 106  To better understand the activities of IpdC, IpdF, EchA20, IpdAB and FadA6, bioinformatic analyses were performed (Table 4.2). Among characterized homologs, IpdF shares 39% amino acid sequence identity with levodione reductase from Corynebacterium aquaticum M-13 [215], which catalyzes the NADH-dependent reduction of a ring ketone. For its part, IpdC shares 30% amino acid sequence identity with FabK from Streptococcus pneumoniae, an enoyl-acyl ACP reductase that catalyzes double bond reduction [216], the reverse of the predicted IpdC reaction. Like FabK, purified IpdCDOC21 contained a flavin (data not shown). Overall, these analyses are consistent with the ability of IpdF and IpdC to catalyze the transformation of 5α-OH HIC-CoA to HIEC-CoA. EchA20 is one of twenty-one EchAs in Mtb. EchAs are members of the crotonase superfamily that are predicted to catalyze the hydration of enoyl-CoAs [217], of which HIEC-CoA, the substrate of EchA20, is an example. A phylogenetic analysis revealed that among Mtb EchAs, only EchA20 clustered with MenB although it shares ~28% amino acid sequence identity, with each of EchA8, EchA18 and MenB. EchA8 and EchA18 are uncharacterized. However, MenB is a 1,4-dihydroxy-2-naphthoyl-CoA synthase that catalyzes an intramolecular Claisen condensation, or Dieckmann cyclization, in menaquinone biosynthesis [218]. This reaction is essentially the reverse of the reaction catalyzed by EchA20. EchA20 also shares 24% amino acid sequence identity with BadI of Rhodopseudomonas palustris (Table 4.2). BadI, a β-ketocyclohexanecarboxyl-CoA hydrolase involved in the anaerobic catabolism of benzoate [219], catalyzes a hydrolytic ring-opening reaction similar to that of EchA20. Overall, the bioinformatics analyses indicate that EchA20 catalyzes the hydrolytic ring-opening of HIEC-CoA to COCHEA-CoA via a reverse Dieckmann cyclization. Nevertheless, it is unclear whether other Mtb EchAs catalyze similar reactions. 107  IpdAB shares 24% amino acid sequence identity with glutaconate CoA transferase (GCT) of Acidaminococcus fermentans [169], a Class I CoA transferase. Most Class I CoA transferases characterized to date catalyze the transfer of CoA between short acyl chains [167, 169, 220, 221]. However, how a CoA transferase is involved in transforming COCHEA-CoA to MOODA-CoA is not immediately apparent. Finally, FadA6 shares 38% amino acid sequence identity with FadA5, a β-ketoacyl-CoA thiolase involved in cholesterol side chain degradation [65] (Table 4.2), consistent with it catalyzing the thiolysis of a COCHEA-CoA ring-opened product containing a β-keto thioester moiety, to MOODA-CoA and acetyl-CoA. 4.3.7 Validation of HIP catabolism using additional mutants  Figure 4.7 Cholesterol-derived metabolite of ΔfadE32 M.smegmatis.  (A) GC/MS traces of culture supernatants of cholesterol-grown ΔfadE32 (red), ΔfadE32 ::msmeg_6015 (blue) and WT (black) M. smegmatis. The major metabolite observed in the mutant was MOODA (inset). (B) GC/MS trace of the product following hydrolysis of metabolite 952 m/z in 1 M NaOH. (C) MOODA purified from ΔfadE32 M. smegmatis incubated with cholesterol  To obtain further evidence for the HIP catabolic pathway suggested by analyses of the ipd mutants and the enzymatic transformations of 5-OH HIC-CoA, the following KstR2-regulated genes were deleted in M. smegmatis MC2155 and the corresponding mutants analyzed : ΔipdF, ΔipdAB, ΔechA20, and ΔfadE32 (msmeg_6011, msmeg_6002-6003, msmeg_6001, and 108  msmeg_6015, respectively). All four mutants were defective for growth on HIP: the ΔfadE32 strain grew more slowly on HIP while the other three did not grow at all (Figure 4.8). The growth defect of each mutant on HIP was complemented by the Mtb or M. smegmatis gene supplied in trans. GC/MS analysis of the culture supernatants revealed that the ipdF and echA20 mutants accumulated low amounts of 5-OH HIC (Figure 4.9). Moreover, ΔfadE32 accumulated a metabolite whose TMS-derivative had an m/z value of 346 (Figure 4.7). The metabolite that accumulated in the supernatant of the cholesterol-grown ΔfadE32 mutant was purified and was identified as MOODA based on 1H, COSY, HMBC, and HSQC-NMR analysis (Appendix C).  The ΔipdAB mutant of M. smegmatis differed from those of RHA1 and Mtb in that it accumulated two major metabolites in the supernatant when incubated with cholesterol. These had m/z values of 254 and 326 when derivatized with TMS (Figure 4.9). The former was purified, and, based on 1H NMR, was identified as an analog of COCHEA lacking the C-10 carboxyl (Appendix C). A similar result was reported in an ipdAB deletion mutant of Comamonas testosteroni [214]. 109   Figure 4.8 Characterization of KstR2 regulon mutants of M. smegmatis. Growth of ΔechA20, ΔfadE32, ΔipdF, and ΔipdAB M. smegmatis mutants on (A) 1.5 mM HIP or (C) 1 mM HIP + 0.2% glycerol. Curves show WT (black), KstR2 regulon mutants (red), and corresponding complements (blue) and are the mean of three biological replicates. (B) CoA metabolomes of mutants. Numbers correspond to CoASH (1), acetyl-CoA (2), HIEC-CoA (3), 5β-OH HIC-CoA (4), 5α-OH HIC-CoA (5), COCHEA-CoA (6), and unknown CoA thioester 992 m/z (7); IS = p-coumaroyl-CoA internal standard. Lighter shaded curves indicate the 768=>261 transition (see Figure 4.4).  The profile of ΔipdF was similar to that of ΔipdC, containing a significant amount of 5-OH HIC-CoA and a lesser amount of 5-OH HIC-CoA (Figure 4.8B). This is consistent with the enzymatic studies inasmuch as neither IpdC nor IpdF alone significantly transformed 5-OH HIC-CoA. The CoA metabolome of ΔechA20 M. smegmatis also contained significant quantities of 110  the 5-OH HIC-CoAs (Figure 4.8B). However, it also contained a small amount of a metabolite whose retention time and m/z value (958) corresponded to that of the transformation product of 5-OH HIC-CoA by IpdC and IpdF (Figure 4.8B). The CoA metabolome of ΔipdAB M. smegmatis was very similar to those of the corresponding RHA1 and Mtb mutants (Figure 4.8B). Finally, the CoA metabolome of ΔfadE32 was indistinguishable from that of WT M. smegmatis. A summary of the data from Sections 4.3.3 – 4.3.7 is provided in Section 6.3.1.  Figure 4.9 Metabolites produced by M. smegmatis strains. GC/MS of culture supernatants of cholesterol-incubated M. smegmatis strains. Cells of the indicated strains were incubated with 0.5 mM cholesterol. Insets show structures of TMS-derivatized 5α-OH HIC and 2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1. * indicates unidentified compounds present in all M. smegmatis extracts that is unrelated to cholesterol catabolism.  4.3.8 HIP-dependent toxicity The failure of the ΔipdAB and ΔipdC mutants to grow on cholesterol (Figure 4.1 and 4.2) is in marked contrast to the phenotype of ΔfadD3 RHA1, which grows on cholesterol to ~50% 111  the yield of wild-type [79]. More specifically, the failure of the ipd mutants to grow on cholesterol despite the fact that the encoded enzymes act downstream of FadD3, suggests that the ipd deletions induce some form of toxicity. To explore this further, KstR2 regulon mutants were grown on a second carbon source in the presence of HIP. Interestingly, the ΔfadE32 and ΔipdF mutants grew on other carbon sources in the presence of HIP (Figure 4.8C), while the ΔipdAB, ΔipdC and ΔechA20 mutants did not (Figure 4.10A, 4.8C, and 4.2D). The inability to catabolize a secondary carbon source in the presence of HIP indicates that there is a HIP-dependent toxicity in some of the mutants, similar to the cholesterol dependent toxicity observed for ipdAB and ipdC mutants described above. One possible form of cholesterol (or HIP)-dependent toxicity is the accumulation of propionyl-CoA, which can be relieved by supplementation with vitamin B12 [49]. However, supplementation of the ΔipdAB, ΔipdC and ΔechA20 mutants with vitamin B12 did not relieve cholesterol-dependent toxicity, indicating that the basis of toxicity is independent of propionyl-CoA in these mutants. However, in analyzing the CoA metabolites of these mutants, the ΔipdAB, ΔipdC and ΔechA20 mutants were noted to contain significantly lower levels (<20%) of CoASH as compared to WT when cells were incubated with cholesterol (Figure 4.10B). In contrast, the ΔfadE32 and ΔipdF mutants contained statistically similar CoASH levels to WT under these conditions. Indeed, the respective levels of CoASH and cholesterol-derived CoA thioesters appeared to be inversely related. For example, when incubated with cholesterol, 5-OH HIC-CoA and COCHEA-CoA accounted for 84 ± 2 and 94 ± 1 % of the total CoA detected in cells of ΔipdC and ΔipdAB RHA1, respectively, indicating that sequestration of CoASH by cholesterol-derived CoA-thioesters may be the basis of toxicity. 112   Figure 4.10 Cholesterol-dependent toxicity. (A) Growth of WT (black), ΔipdAB (red), and ΔipdAB::ipdAB (blue) Mtb grown on 7H9 media containing 0.5 mM cholesterol and 0.2% glycerol. Data represent the average of biological triplicates. (B) The relative abundance of CoASH (768261) was normalized to the internal standard (p-coumaroyl-CoA (914407)) in KstR2 regulon mutants. * indicates p < 0.05 as compared to WT strain. Error bars represent standard deviation. The number of replicates, N, was: 5, 5, 5, and 3 for WT, ΔipdAB, ΔipdC, and ΔfadD3 ΔipdAB RHA1, respectively; 2, 1, and 1 for WT, ΔipdAB, and ΔipdC Mtb, respectively; and 4, 5, 1, 4, and 1 for WT, ΔipdAB, ΔechA20, ΔipdF, and ΔfadE32 M. smegmatis, respectively.    113   Structural and mechanistic characterization of IpdAB Chapter 5:5.1 Introduction Elucidation of the catabolism of cholesterol Rings C and D (Chapter 4) implicated IpdAB in the opening of Ring C. This result was unexpected as apparent hydrolytic cleavage of a carbon-carbon bond is unprecedented within the Class I CoA transferase family to which IpdAB belongs. IpdAB is of specific interest due to its role in pathogenesis. Mtb deficient in ipdAB were unable to grow in macrophages (Section 4.3.2) and Rhodococcus equi deficient in ipdAB and ipdA2B2 is patented as a live vaccine for use in horses against pyogranulomatous pneumonia [23].  Herein, a combination of x-ray crystallography, steady-state kinetics, directed-mutagenesis, and isotopic labelling was employed to characterize the function of IpdAB from Mtb and RHA1. The data distinguishes IpdAB from Class I CoA transferases and indicate that IpdAB opens the last steroid ring of cholesterol by acting as a hydrolase. Overall, this study identifies a novel catalytic function within the CoT superfamily and provides important insights into a virulence determinant and potential target for novel anti-tuberculosis therapeutics.   Experiments pertaining to data collection,structure refinement, and structural analyses of the IpdAB X-ray crystal structures were performed by Sean Workman, Dr. Liam Worrall, and Dr. Nobuhiko Watanabe from the Strynadka Lab at UBC. Some LC-MS data was acquired by Dr. Israël Casabon. The contents of this chapter were prepared as a manuscript for submission in 2018 as follows:  Crowe, A.M, Workman, S.,Worrall, L., Watanabe, N., Strynadka, N., and Eltis, L.D. (2018). IpdAB, a virulence factor in Mycobacterium tuberculosis, is a cholesterol ring-cleaving hydrolase.  114  5.2 Materials and methods 5.2.1 Chemicals and reagents CoASH, NAD+, FMN, sodium acetate, sodium propionate, sodium succinate, acetyl anhydride, propionic anhydride, succinyl anhydride, acetoacetyl-CoA, and acetyl-CoA were purchased from Sigma- Aldrich. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. Phusion DNA polymerase and Taq DNA ligase was purchased from Thermo Scientific. Oligonucleotides were purchased from Integrated DNA Technologies (IDT). 5αOH-HIC was provided by Victor Snieckus (Queen’s University, Kingston, Canada). All other reagents were of HPLC or analytical grade. Buffers and solvents were prepared as previously described in Section 4.2. 5.2.2 Bioinformatic anaylsis of CoA transferases Amino acid sequences of CoTs and IpdAB homologs were aligned using TCOFFEE- ESPRESSO [183]. For homomeric enzymes containing domains complementary to α and β subunits, the domains were treated separately. Phylogenetic trees were generated using Approximate Likelihood-Ratio Test (aLRT) via PhyML on the phylogeny.fr server [182]. 5.2.3 DNA manipulation and plasmid construction: DNA was propagated, amplified, digested, ligated, and transformed using standard protocols [180]. Oligonucleotide- directed mutagenesis was performed using the QuikChangeTM PCR protocol with slight modifications. Briefly, a single 5’ phosphorylated mutagenic DNA oligomer was annealed to pTipR1IpdAB then amplified using Phusion DNA polymerase. Taq DNA ligase was added to generate a single stranded mutagenized plasmid. DpnI was used to remove template DNA and the remaining ssDNA was transformed into E. coli NovaBlue. pTipR1IpdAB variants E58BA, E105AA, E105AD, R92BM, and R126BM were generated using 115  the following respective oligonucleotides (substituted nucleotides are underlined): 5’-pCTGATCACCGACGGTGCGGCCCTGATCTTCGCG-3’, 5’ pGTCCGCGAAATGGACGCG-GGCATGGTCAAGTGC-3’, 5’-pGTCCGCGAAATGGACGACGGCATGGTCAAGTGC-3’, 5’-pTCGCCTCCGGCCGGATGCACGTGGTGATGG-3’, and 5’-pCAGATGTTCGGCGTC-ATGGGCGCACCCGGCAAC-3’. The nucleotide sequence of variants was confirmed.  5.2.4 Production of COCHEA-CoA: COCHEA-CoA was produced enzymatically from 5αOH-HIC-CoA as described in Section 4.2.1.1 with the following variations. Following synthesis of 5αOH-HIC-CoA, the reaction mixture was loaded onto 100 mg prepacked 2-(2-pyridyl)ethyl-functionalized silica (Supelco, 54127-U), washed with acetonitrile: isopropanol: water: acetic acid (9:3:4:4 v:v:v:v) to remove unreacted precursors, then eluted in 20 mM ammonium acetate (pH 7.0) in 80% methanol. Methanol was removed under nitrogen. Four μmoles of 5αOH-HIC-CoA were added to a mixture containing IpdCDOC21 (5 μM), IpdF (5 μM), EchA20RHA1 (5 μM), IpdABRHA1 (1 μM), 1 mM NAD+, and 10 μM FMN in 50 mM sodium phosphate, pH 8.0. The reaction mixture was incubated at 30oC until completion as determined using HPLC (~3 h). Proteins were precipitated using methanol and the reaction mixture was lyophilized overnight then suspended in 500 μl water. COCHEA-CoA (tR = 6.1 min) was purified using an HP1100 series HPLC (Agilent Technologies) equipped with a Luna 3u PFP(2) 50 x 4.6 mm column (Phenomenex) operated at 1 ml min-1 and separated over a gradient of 20.0 – 30.2% methanol (90%) in 100 mM ammonium acetate, pH 4.5. HPLC- purified fractions of COCHEA-CoA were pooled, lyophilized, and then suspended in 400 μl water. To desalt, COCHEA-CoA was loaded onto an HPLC equipped with the PFP(2) column, washed for 2 min with water + 0.1% acetic acid then 116  eluted using 100% methanol. Typical mole yields of 25% were observed from racemic 5αOH-HIC-CoA 5.2.5 Protein production and purification: IpdABRHA1 and variants were produced using RHA1 as a host strain as described in Section 4.2.3 with the following modifications. Following nickel affinity chromatography, IpdABRHA1 and its variants were dialyzed overnight against 25 mM HEPES, pH 7.5, 50 mM NaCl then concentrated to ~5 mg ml-1 to which 1:1000 (mol:mol) α-thrombin was added and allowed to digest at room temperature for up to 20 h until complete as determined using SDS PAGE. Digested IpdABRHA1 was loaded onto a 10/100 MonoQ anionic exchange column (GE Healthcare) and purified using an AKTA Purifier (GE Healthcare) operated at 2 ml min-1 in 25 mM HEPES, pH 7.0 using a gradient of 150 – 400 mM NaCl. IpdABRHA1 eluting at a conductance of ~36 mS/cm then exchanged into 25 mM HEPES, pH 7.5, 50 mM NaCl, and concentrated to ~15 mg ml-1 using a 10K Amicon centrifugation unit (Millipore). Enzyme was flash frozen in liquid nitrogen as beads and stored at -80 C until further use.  5.2.6 Characterization of IpdAB and variants: Circular dichroic (CD) spectra were recorded at room temperature using a Jasco model J-810 spectropolarimeter. Far-UV CD spectra (190-250 nm) were recorded using a 1 mm quartz cuvette containing 3 μM protein in 10 mM sodium phosphate, pH 8.0. Cuvettes were continuously purged with nitrogen during spectra collection. Spectra were recorded in triplicate and averaged. Size exclusion chromatography multiangle light scattering (SEC-MALS) data were obtained using a HPLC 1260 Infinity LC (Agilent Technologies) coupled to a Superdex 200 5/150 column (GE Healthcare). Data were collected using a miniDAWN TREOS multiangle 117  static light scattering device and an Optilab T-rEX refractive index detector (Wyatt Technologies). Sample of 25 μl containing 0.5 mg ml-1 protein were analyzed. The HPLC was operated at 0.25 ml min-1 in 25 mM HEPES, pH 7.5, 50 mM NaCl. Molecular weights of complexes were calculated using the ASTRA6 program (Wyatt Technologies).  Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay with bovine serum albumin as a standard. 5.2.7 Crystallization of IpdAB and variant Crystals of IpdABRHA1were obtained by sitting-drop vapour diffusion using a reservoir solution of 1.9 M ammonium sulphate and 0.2 M sodium potassium tartrate. Drops contained 1 μl of protein solution (10 mg ml-1) mixed with an equal volume of reservoir solution and were incubated at room temperature. Single clear bipyramidal crystals were observed within 7 d for WT and 60 d for the E105AA mutant. To generate substrate-bound structures, 5 μl of ~10 mM COCHEA-CoA in 2.4 M ammonium sulphate, 0.2 M sodium potassium tartrate was added directly to protein crystals in the drop and allowed to soak for 24-72 h at room temperature. 5.2.8 Crystallographic analysis and refinement: Single IpdAB crystals were looped and flash-frozen in liquid N2 without additional cryoprotectant. Diffraction data for substrate-free IpdABRHA1 and the E105A·COCHEA-CoA complex were collected at Beamline 08B1-1 at the Canadian Light Source in Saskatoon, Saskatchewan. Diffraction data for IpdABRHA1 WT•COCHEA-CoA complex were collected at Beamline 5.0.2 at the Advanced Light Source in Berkeley, California. All data were processed using Xia2 [222], XDS [223], and Aimless [224]. The native structure was solved by molecular replacement using Phaser [225] using a starting model based on the backbone atoms of coordinate set PDB#: 1POI. Enzyme•substrate complexes were grown isomorphously and 118  determined using difference Fourier analysis. Iterative cycles of model-building and refinement were performed with Coot [199] and Phenix [198]. For the IpdABRHA1 WT•COCHEA-CoA complex, a feature enhanced map [226] was used to guide the placement of ligand in the active site. Active site volume for IpdABRHA1 and GCT (PDB# 1POI) was measured using the CASTp server [204] and a 2.0 Å probe. 5.2.9 In vitro activity of IpdAB The transformation of various acyl-CoAs was performed in 100 μl 10 mM sodium phosphate, pH 8.0 containing 1 μM IpdABRHA1, 100 μM COCHEA-CoA, 125 μM CoASH and, as appropriate 5 μM FadA6 from Mtb. Reactions were incubated at room temperature for 1 h and terminated with the addition of 200 μl MeOH (0.5 % acetic acid). To test CoA transferase activity, the reactions contained either 100 μM acetyl-CoA, propionyl-CoA, succinyl-CoA, COCHEA-CoA, or MOODA-CoA and 100 μM each of acetate, propionate, succinate and MOODA. Reaction products were analyzed using a HP1100 Series HPLC attached to a 4.6 x 50 mm Luna 3u PFP(2) column operated as described in Section 4.2.1. 5.2.9.1 Steady-state kinetic characterization of IpdAB: Steady- state kinetic parameters were evaluated using spectrophotometric assays recorded on a Cary 5K UV-Vis-NIR spectrophotometer (Agilent Technologies). The FadA6-catalyzed thiolysis of acetoacetyl-CoA was determined using 0.1 μM FadA6, 100 μM CoASH, and 50-600 μM acetoacetyl-CoA in 200 μl HEPES, pH 7.5, 10 mM MgCl2 (I= 0.05 M) at 25oC by following the decrease in absorbance at 303 nm due to loss of the acetoacetyl-CoA-Mg2+ enolate (ε = 16.9 mM-1cm-1 [47]) as previously described [65]. The IpdAB-catalyzed hydrolysis of COCHEA-CoA was followed using a coupled reaction with FadA6 by following the consumption of 119  COCHEA-CoA at 252 nm (ε = 17.2 mM-1 cm-1). Reactions were performed in 200 μl HEPES, pH 7.5, 1 mM MgCl2 (I= 0.01 M) at 25oC containing 50 μM CoASH, 5 μM FadA6, 0.01 μM IpdAB, and 20-110 μM COCHEA-CoA. The specific activity of IpdABRHA1 variants was determined using 2 μM FadA6, 50 μM COCHEA-CoA, 50 μM CoASH, and 0.01 – 3.5 μM IpdAB variant. The extinction coefficient of COCHEA-CoA was empirically derived by measuring the decrease in absorbance at 252 nm upon complete conversion to MOODA-CoA (ε310nm = 11.9 mM-1cm-1). Steady-state kinetic parameters were determined by least fit squares fitting of the Michaelis-Menten equation to the data using the GraphPadTM analysis software.  5.2.10 Structure assignment for MOODA-CoA: The location of the CoA moiety in MOODA-CoA was determined using NMR. 1 μmol COCHEA-CoA was incubated with 2 μM IpdAB, 10 μM FadA6, and 2 μM CoASH in 10 mM sodium phosphate, pH 8.0 at room temperature. Upon completion, the proteins were removed using methanol and the reactions were lyophilized overnight. The resulting residue was suspended and dried twice in 500 μl deuterated methanol (D-MeOD) then suspended in 400 μl D2O. 1H-NMR, 1H-1H COSY, 1H-1H TOCSY, and 1H-13C HMBC spectra were collected on a Bruker 600 MHz spectrophotometer. The sample contained MOODA-CoA (1.0 mM), acetyl-CoA (1.3 mM), and CoASH (1.3 mM). NMR data were analyzed using the Academic ACD NMR Processor Software (ACD Labs). Spectra of MOODA-CoA were compared with those recorded for MOODA. 5.2.11 Attempts to trap acyl-enzyme intermediates Fifty μM COCHEA-CoA and 50 μM IpdABRHA1 were incubated in 50 μl of 10 mM sodium phosphate, pH 8.0 for 5 min then sodium borohydride was added to a final concentration 120  of 20 mM. After an additional 30 min of incubation, samples were thoroughly desalted and exchanged into water using a 10 kDa Centricon centrifugation unit (EMD Millipore). The molecular weight of intact protein samples was determined using a Waters Xevo G2 qTOF operated by The University of British Columbia Proteomic Core Facility. The specific activity towards COCHEA-CoA was tested for the desalted IpdABRHA1 as described above for the IpdAB variants.  5.2.12 KD determination for IpdAB E105AA: The dissociation constant (KD) of IpdAB E105AA for COCHEA-CoA was determined by following the increase in absorbance at 310 nm upon formation of the complex. IpdAB E105AA (5 μM) was titrated with 0-5.2 μM COCHEA-CoA in 200 μl HEPES, pH 7.5, 1 mM MgCl2 (I=0.01 M) at 25oC. The reference cuvette contained 200 μl HEPES, pH 7.5, 1 mM MgCl2 (I=0.01 M) at 25oC. Difference spectra were recorded after each addition of substrate to the two cuvettes. Data used for calculation represent the difference between titrations of COCHEA-CoA into IpdAB E105AA or assay buffer alone. The instrument was blanked using the assay buffer. Steady-state kinetic parameters and dissociation constants (KD) were determined by least fit squares fitting of the Michaelis-Menten equation or quadratic binding equation (Eq. 1, Section 2.2.9) to their respective data sets using the GraphPadTM analysis software.       5.2.13 Deuterium incorporation into COCHEA-CoA: 100 μM COCHEA-CoA was incubated for 30 min with and without 2.5 μM IpdAB in 50 μl of 10 mM sodium phosphate, pH 8.0 prepared in D2O. Reactions were terminated by 5 μl of acetic acid. Samples were centrifuged to remove precipitated protein then filtered 0.45 μm, diluted 1:49 in water and analyzed by LC-MS/MS and/or LC-MS3.   121  5.2.14 Proton-deuterium exchange NMR experiments: Proton-deuterium exchange experiments were performed in 400 μl 10 mM sodium phosphate, pH 8.0 on a Bruker 500 MHz spectrophotometer.  Before use, IpdABRHA1 was exchanged into 10 mM sodium phosphate, pH 8.0 in D2O using a Nanosep 10K Centrifugal Device (PALL Life Sciences). 1H-NMR spectra of 1.6 mM COCHEA-CoA were recorded before and after addition of 50 nM IpdAB every minute for 45 minutes. A delay of 5 minutes occurred between the addition of IpdAB and the first recorded spectra. Aliquots of the reaction at the start and end of the experiments were analyzed by LC-MS to confirm incorporation of deuterium into COCHEA-CoA. A time course plot of relative proton integration was generated by normalizing each proton integration with the C-37 methyl group in the Coenzyme A moiety. The initial rate of deuterium exchange at C-9 of COCHEA-CoA was calculated using a least fit squares of Equation 3 to the first 10 minutes of the progress curve as performed by GraphPad. [S] was calculated using Equation 4 where [S]o, Ha(t) and H0.91(t)  represent the initial substrate concentration and the integration of proton (a) or C-37 at time t, respectively.   Equation 3:  ௗሾௌሿௗ௧ ~ െ ݇ሾܵሿሾܧሿ Equation 4: ሾܵሿ ൌ ሾܵሿ௢ ுೌሺ௧ሻுబ.వభሺ௧ሻ 5.2.15 18O labelling of COCHEA-CoA, MOODA-CoA, and IpdAB 100 μM COCHEA-CoA was incubated with and without 5 μM IpdABRHA1 (or 5 μM IpdAB, 20 μM FadA6, and 125 μM CoASH in MOODA-CoA experiments) in 50 μl of 10 mM sodium phosphate, pH 8.0 repaired in 97% 18[O] H2O (Sigma Aldrich) for 15 minutes at room temperature. Reactions were terminated with the addition of 2 μl acetic acid or 200 μl methanol. Samples were ultracentrifuged  (16,000 g, 5 min) to remove protein, filtered 0.45 μm through 122  PTFE, then immediately frozen in liquid nitrogen and stored at -80oC until analysis by LC-MS/MS. To look at 18O labelling of IpdAB, the reactions were performed as described above; however no acetic acid or methanol was added. IpdAB was desalted into water using a 10K centrifugation unit (Millipore) then subjected to intact protein MS as described above.  5.2.16 Mass spectrometry: Mass spectra were recorded using a Qstar mass spectrometer (Agilent Technologies) coupled to an HP 1100 series HPLC (Agilent Technologies) equipped with a 50 x 0.3 mm C18(2) column operated at in positive ion mode (Ion spray voltage= +5500V, Ion source temperature= 350oC). CoA thioesters were eluted using a gradient of 100 mM ammonium formate, pH 3.5 into acetonitrile + 0.1% formic acid at a flow rate of 4 μl min-1. High resolution MS3 of CoA thioesters were analyzed on a Bruker Impact-II Q-ToF equipped with a 150 x 0.25 mm Luna 3 μm PFP(2) (Phenomenex) column as describedin Section 4.2. CoA thioesters were eluted using a gradient of 100 mM ammonium acetate in 2% methanol and 20 mM ammonium acetate in 98% methanol. Mass spectrometers were calibrated daily.  123  5.3 Results 5.3.1 Phylogenetic analysis of IpdAB and Class I and II CoA transferases  Figure 5.1 Bioinformatic analysis of IpdAB and homologs. Phylogenetic trees displaying IpdA and α-subunits (A) or IpdB and β-subunits (B) from Class I and II CoA transferases. Shaded regions indicate Gram positive IpdA or IpdB (blue), Gram negative IpdA or IpdB (green), or Class I β-keto-CoA (purple), Class I (yellow), and Class II (grey) CoA Transferases. Proteins displayed are IpdA from R.jostii RHA1 (IpdARHA1), R. equi (IpdAR.equi; IpdA2R.equi), M. smegmatis (IpdAM.smeg ), M. tuberculosis (IpdAMtb), Steroidobacter denitrificans (IpdAACG33), and Comamonas testosteroni CNB-2 (IpdACNB-2); β-ketoadipyl-CoA transferase from Pseudomonas putida (PcaI), Glutaconate CoA transferase from Acidaminococcus fermentans (GCT), Citrate Lyase from Enterobacter aerogenes (CitC), Citrate Lyase from Clostridium argentinense (CitF), Butyrate-acetoacetate CoA transferase from Clostridium acetobutylicum (CtfA), Acetate CoA transferase from Escherichia coli (ACT), Succinyl-CoA transferase from Bacillus subtillus (ScoAB.sub), Succinyl-CoA transferase from Helicobacter pylori (ScoAH.pylori), Succinyl-CoA transfease from pig heart, Propionyl-CoA transferase from Clostridium propionicum (PCT), and Acetate CoA transferase from Escherichia coli (YdiF). Additional information is available in the Supplemental Information (SI). (C) Amino acid alignment of IpdB from CNB-2, Mtb, and RHA1 with well characterized Type I CoA transferases. Red box indicates location of highly conserved catalytic glutamic acid in Class I CoA transferases. Numbering corresponds to residue number in IpdBRHA1 (D) Conservation of active site glutamate in IpdAs.   124  To better understand the relationship of IpdAB to CoTs, the phylogeny of IpdA and IpdB with respect to the α and β subunits, respectively, of characterized Class I and II CoTs was analyzed. Sequence alignments were structure-guided and only sequences corresponding to the conserved core domains of the CoTs were used in the phylogenetic analysis to minimize biases resulting from insertions (Figure 5.1). This analysis revealed that IpdA homologs from steroid-degrading bacteria formed a clade distinct from each of the three formed by the α subunits of the Class I CoTs, Class I β-keto-CoTs and Class II CoTs, respectively (Figure 5.1). IpdAs clustered most closely with the heterotetrameric Class I β-keto- CoTs and, within the IpdA clade, orthologs from actinobacteria (blue) and proteobacteria (green) formed subclades. Analysis of IpdBs with the CoT β subunits revealed similar relationships except that PcaI, the β subunit of the β-ketoadipate:succinyl-CoA transferase PcaIJ, clustered differently than the α subunit, PcaJ (Figure 5.1).  5.3.2 Characterization of IpdAB To biochemically and structurally characterize IpdAB, IpdABMtb and IpdABRHA1 were overproduced and purified to apparent homogeneity using metal affinity chromatography. Both orthologs were eluted as colourless preparations. RHA1 was a better expression host than E. coli, although IpdABRHA1 was produced to higher levels than IpdABMtb and was more stable once purified. SEC MALS analyses revealed that IpdABRHA1 had a molecular weight of 109.7 ± 0.1 kDa, corresponding to an α2β2 heterotetramer as observed in Class I CoTs.   125  5.3.2.1 In vitro transformations and steady state kinetics All CoTs characterized to date catalyze the inter- or intra-molecular transfer of CoA from a CoA thioester to a free carboxylate [167, 169]. To test whether IpdABRHA1 had similar activity, the enzyme was incubated with a variety of CoA donors and small organic acids. HPLC analysis failed to detect the transfer of CoA from any of acetyl-CoA, propionyl-CoA and succinyl-CoA to acetate, propionate, or succinate (data not shown). Additionally, IpdAB did not detectably catalyze the hydrolysis of CoA donors (data not shown).   Figure 5.2 In vitro activity of IpdAB. (A) HPLC trace of in vitro transformation of COCHEA-CoA with IpdABRHA1 and FadA6. 100 μM COCHEA-CoA was incubated with 1 μM IpdABRHA1/ IpdABMtb, 5 μM FadA6, and/or 125 μM CoASH in 10 mM sodium phosphate pH 8.0. (B) 13C – 1H HMBC NMR spectral overlay (0-3 ppm 1H; 180-230 ppm 13C) of MOODA and MOODA-CoA. Spectra were recorded on a 600 MHz spectrophotometer in D2O. (C) Steady state analysis of FadA6 and 126  acetoacetyl-CoA (AcAcCoA). (D) Effect on the ratio between IpdAB and FadA6 on in vitro turnover of COCHEA-CoA to MOODA-CoA. (E) Steady state analysis of IpdAB and FadA6 towards COCHEA-CoA. Curves for (C, D, and E) indicate best squares least fit of Michaelis-Menten equation to the data. Data was obtained in 200 μl HEPES pH 7.5 (I= 0.01 M) at 25oC. (F) Diagram outlining the proposed reaction catalyzed by IpdAB and FadA6. Carbon numbering used throughout this thesis are identified.  Table 5.1 Steady state parameters for FadA6 and IpdAB Enzyme Substrate kcat (s-1)KM (μM) kcat/KM  (× 104 M-1s-1)FadA6Mtb AcAcCoA 4.8 (0.8) 300 (100) 1.6 (0.8) IpdABRHA1 COCHEA-CoA 5.8 (0.8) 120 (40) 5 (2)   5.3.3 IpdAB catalyzed the efficient transformation of COCHEA-CoA Incubation of COCHEA-CoA with IpdAB and a thiolase yielded a metabolite presumed to be MOODA-CoA (Section 4.3.5). To extend this observation, the ability of IpdAB to transform COCHEA-CoA and other CoA thioesters was tested in vitro. In the presence of a thiolase, either FadA5 or FadA6 from Mtb, and CoASH, both IpdABMtb and IpdABRHA1 stoichiometrically transformed COCHEA-CoA into acetyl-CoA and a second CoA thioester of 952 m/z, consistent with MOODA-CoA (Figure 5.2A). No compounds other than COCHEA-CoA, MOODA-CoA, acetyl-CoA, and CoASH were detected using LC-MS. Neither IpdAB nor a thiolase alone detectably transformed COCHEA-CoA. Since IpdABMtb and IpdABRHA1 displayed similar activities, subsequent studies were performed using the more stable ortholog, IpdABRHA1. To evaluate the efficiency of the IpdAB reaction, a coupled assay with IpdABRHA1 and FadA6 was established. The thiolytic activity of FadA6 towards β-keto CoA thioesters was first evaluated using acetoacetyl-CoA by following the decrease in absorbance at 310 nm from the 127  acetoacetyl-CoA-Mg2+ enolate. The steady-state kinetic parameters indicated that FadA6 readily cleaves acetoacetyl-CoA (Table 5.1). In a coupled assay with IpdAB, the rate of COCHEA-CoA consumption, as measured spectrophotometrically by the decrease in absorbance at 252 nm, was limited by FadA6 at mole ratios below 400:1 (FadA6:IpdAB; Figure 5.2C). The product of the IpdAB reaction was turned over by FadA6 at a rate of 0.014 ± 0.006 s-1. In the presence of saturating amounts of FadA6, IpdAB had a relatively high specificity constant for COCHEA-CoA (kcat/KM = 5 × 104 M-1s-1; Figure 5.2D, Table 5.1). Similar results were obtained using FadA5 instead of FadA6 (data not shown). NMR was used to definitively identify the second CoA thioester produced in the enzymatic transformation of COCHEA-CoA as MOODA-CoA in which the CoA moiety is attached at carbon-8 (C-8) of MOODA. More specifically, comparison of the 1H-13C HMBC spectra of MOODA-CoA and MOODA revealed a ~23 ppm increase in the chemical shift for C-8 in MOODA-CoA as compared to MOODA (Figure 5.2B). Given that FadA5 and FadA6, as well as all β-keto-CoA thiolases characterized to date, act on β-keto-CoA thioesters, the location of the CoA moiety on MOODA-CoA indicates that the substrate for FadA6 is 6-methyl-3,7-dioxodecanedioyl-CoA (MeDODA-CoA) (Figure 5.2F). Failure to observe this species in the absence of FadA6 may be because the equilibrium lies far to the left.  128  5.3.4 The structural fold of IpdAB is typical of Class I CoTs  Figure 5.3 Structure of IpdABRHA1 (A) Biological assembly of IpdABRHA1. IpdA and IpdB subunits are depicted in blue and orange, respectively. COCHEA-CoA (lactonized) is displayed in the active sites.  (B) Surface topology of the IpdABRHA1 (left) and GCT (right) active site pockets. The location of the catalytic Glutamate is displayed in red. (C) Location of candidates for the catalytic acidic residue in the β-subunit from structural alignments. Ordered water molecule is shown as a red dot. (D) Structural overlay of the IpdABRHA1 and GCT (grey) active sites including catalytically relevant residues.   To further characterize IpdAB, the X-ray crystallographic structure of IpdABRHA1 was solved to 1.7 Å resolution. The asymmetric unit of the P43212 IpdABRHA1 crystals contained a single αβ heterodimer. The α2β2 assembly, observed in solution, was formed from two αβ protomers related via a two-fold axis of symmetry in the crystal lattice (Figure 5.3A). Ordered electron density for all the residues of IpdAB was observed except for the N terminal methionine of IpdA, which was removed in cleaving the affinity tag, and the first six residues of IpdB. Data collection and structure refinement statistics are presented in Appendix B. 129   IpdAB possesses the same core fold as Class I CoTs. More specifically, the core of IpdA consists of a seven-stranded parallel β-sheet sandwiched between helices α1-4 on one side and α5-7 on the other. The core of IpdB is similar except that the β-sheet is six-stranded (five parallel strands, one anti-parallel) and is sandwiched between fewer helices (α1-2 and α5-6, respectively). IpdA is further distinguished from IpdB by a 13-residue loop consisting of Thr144-Thr156 that overlaps the second IpdA subunit (denoted by A’) and likely stabilizes the tetrameric assembly via two hydrogen bonds: one between Tyr147A (Oη) and Arg141A’ (peptide N), and a second between Arg141A (Nη) and Pro146A’ (peptide O). Consistent with the phylogenetic analyses, the structural fold of IpdAB closely resembles that of GCT (rmsd on 371 shared Cα with GCT, PDB# 1POI = 2.39 Å). 5.3.5 IpdAB has distinct active site residues Each IpdABRHA1 protomer harbours a single, large active site pocket located at the interface between the two subunits. More specifically, the pocket is ~3300 Å3 and lies between helix α5 of IpdA, helices α3,4 of IpdB, and the β-sheet of IpdB. A channel of ~28 Å projects out of the active site and follows the contours of the interface between the two subunits. The active site pocket of IpdAB is approximately two times larger than that of GCT (1780 Å3) and is much less solvent-exposed (Figure 5.3B). These differences reflect IpdB’s active site loop which, at 6 residues (Thr55B to Leu60B), is 18 residues shorter than that of GCT. The corresponding loop in GCT also carries Glu54β, the key catalytic residue of Class I CoTs [169]. The IpdB loop carries two acidic residues, Asp56B and Glu58B. However, the Cδ of Glu58B carboxylate is located ~13 Å away from the Cδ of Glu54B in GCT when superimposed with IpdAB and appears to stabilize the protomer by forming a water mediated hydrogen bond with Arg32A  (Figure 5.3C). Asp56B appears to contribute to the stability of the active site loop via hydrogen bonds to the peptide 130  nitrogen of Ala59B (Figure 5.3C), and is not orientated towards the substrate-binding pocket, suggesting it does not have a role in catalysis. Nevertheless, IpdAB does contain an acidic residue at the center of its active site: Glu105A (Figure 5.3D). Inspection of the sequence alignments used for the phylogenetic analyses revealed that Glu105A is conserved in IpdAB orthologs but not in Class I CoTs (Figure 5.1D). By contrast, the typical catalytic base, Glu54β, of Class I CoTs does not occur in any of the IpdAB orthologs (Figure 5.1C).  Figure 5.4 Structure of IpdABCOCHEA-CoA. (A) Lactonized COCHEA-CoA binds along a long channel between IpdA (blue) and IpdB (orange). Predicted CoA moiety binding residues are shown. (B,C) Active site of IpdABRHA1 E105AA∙COCHEA-CoA (B) and WT∙COCHEA-CoA (C) displaying predicted catalytically relevant residues. Shown are the respective Fo-FC maps (green mesh) around the substrate contoured at 2.0 σ. Location of proposed catalytic water in the absence of lactonized COCHEA-CoA is displayed as a white dot. (D) Diagram illustrating residues predicted to make contacts with the lactonized COCHEA-CoA in the IpdABRHA1 active site.  131  5.3.6 Structure of IpdAB·COCHEA-CoA complexes To gain more insight into the function of IpdAB, structures of IpdABRHA1 WT and E105AA soaked with COCHEA-CoA were solved to 1.6 and 1.4 Å, respectively. The location of the thioester sulphur atom in the WT∙COCHEA-CoA complex was determined using anomalous scattering. Refinement data are summarized in Appendix B. Structures of substrate-bound IpdABRHA1 were remarkably similar to the substrate-free IpdABRHA1 structure (rmsd on 462 shared Cα= 0.2 Å). The few notable differences include a displacement of the sidechain atoms of Arg126B and Arg120B by 3.3 and 1.0 Å, respectively, presumably to accommodate the substrate, and the movement of the side chain atoms of Arg147B and Tyr85A towards the substrate by 4.0 and 1.0 Å, respectively. With the exception of the acyl moiety in the active site and the substituted residue, the WT and E105AA substrate-bound structures were essentially indistinguishable (rmsd of 0.07 Å on 489 common Cα atoms). In both IpdAB∙COCHEA-CoA complexes, the substrate is bound in a channel between the subunits with the CoA adenine binding ~21 Å away from the CoA thioester sulfur atom in the active site (Figure 5.4). This is similar to what has been reported in Class I CoTs [147]. For example, superposition of the IpdAB∙COCHEA-CoA complexes with those of YdiF from E.coli (PDB# 2AHV) and pig heart SCOT (PDB# 3OXO) revealed that their respective CoA sulphur atoms are within  1.2 Å of each other. Nevertheless, the adenine-binding pocket of IpdAB is located ~7 Å from the position where it is found in CoTs. Notable interactions with the CoA moiety include coordination of the diphosphate group by the side chains of Arg120B, Asn99B, and Tyr85A; π-π stacking between the adenine group and Phe112B; and coordination of the adenine amino group by Thr119B (Figure 5.4A).  132  Electron density in the active site of the E105AA·COCHEA-CoA complex clearly revealed a 5-membered lactone ring generated from the cyclization of the C-1 carboxylate to C-4 (Figure 5.4B). The cyclohexenone ring of the bound COCHEA-CoA is largely planar with the C-8 oxo and C-11 methyl group orientated 180o from each other. The lactone ring is orientated perpendicularly to the cyclohexanone ring such that C-4 is an (S) stereocenter. No electron density was observed corresponding to an (R) C-4 stereocenter (Figure 5.4B). Torsional angles about C-4 deviate slightly from an optimal sp3 hybridized center due to a slight bend of the lactone ring away from the C-11 methyl resulting in angles of 104 and 110o for O-1/C-4/C-1 and O-1/C-4/C-5 bonds, respectively. Similarly, the C-8 oxo is bent slightly (Φ=116o) towards Arg92B and forms hydrogen bonds with that residue (2.9 Å) and Asn131B (2.8 Å) (Figure 5.4B). The C-11 methyl group sits in a hydrophobic pocket formed by Phe80B, Phe84B, Val83B and Trp29A. The C-1 ester carbonyl hydrogen bonds (3.1 Å) to an ordered water molecule coordinated by Arg126B, Arg32A, and Asp80A. The C-10 thioester carbonyl orients towards the Nη of Arg126B at a distance of 4.0 Å.  Electron density in the active site of the WT IpdABRHA1·COCHEA-CoA complex was less resolved than in the E105AA·COCHEA-CoA complex. However, additional residual density outside of the active site fit the CoA moiety well, suggesting that the substrate may bind in more than one conformation (Figure 5.4C). Using a feature enhanced map [226], density for the 5-membered lactone observed in the E105AA structure could be clearly identified. No density for the C-1 carboxylate was observed indicating that the un-lactonized form of COCHEA-CoA was not present. Comparison of the lactonized COCHEA-CoA from the two structures illustrates distinct differences (Figure 5.4B,C). First, the cyclohexanone ring is not planar in the WT complex: the C-8 oxo is bent upwards towards Arg92B. Second, the C-8 oxo and C-10 thioester 133  are also not planar, suggesting C-9 is sp3 hybridized and protonated. Third, the lactone exhibits bond torsion between C-1/C-2/C-3 (Φ=96o) deviating from the predicted 105o. Lastly, the lactone in the WT structure is rotated ~45o away from its location in the E105AA complex, presumably to avoid steric clash with Glu105A. Interestingly, the Oε of Glu105A forms a 2.8 Å hydrogen bond with the Nε proton of Arg126B positioning the Glu105A carboxylate directly under C-4 of COCHEA-CoA (Figure 5.4C). Presumably, in the absence of substrate lactonization, a catalytic water molecule could be accommodated between C-4 of COCHEA-CoA and Glu105A (Figure 5.4C, white circle).  In neither complex is there any evidence of an oxyanion hole to accommodate the CoA thioester oxo of the bound COCHEA-CoA. By contrast, an oxyanion hole appears to stabilize the thioester oxo in YdiF and SCOT [175].  5.3.7 Identification of catalytically essential residues The structural data indicate direct interactions between the acyl-moiety of COCHEA-CoA and each of the side chains of Arg92B, Glu105A and Arg126B such that they may potentially activate the substrate during catalysis. Glu58B on the other hand is located away from the bound substrate and thus predicted to be non-essential for catalysis. Importantly, these residues are all conserved in IpdABs (Figure 5.1C). To test the catalytic relevance of these residues, each was substituted and the variants purified. CD spectroscopy and SEC MALS indicated that all variants had the same global secondary and tertiary structures as WT IpdAB (Figure 5.5). The E58BA variant showed only a minor reduction in specific activity compared to WT, consistent with its localization and a non-catalytic role. In contrast, the other variants (E105AA, E105DA, R92MB, and R126MB) had no detectable activity, consistent with these residues playing an essential role in catalysis (Table 5.2). 134   Figure 5.5 Characterization of IpdAB (A) SDS-PAGE showing ~1 μg of indicated protein (B) SEC-MALS analysis of IpdAB and variants. Shown is the absorbance at 280 nm and the calculated molecular weight by the ASTRA6 software of proteins eluting from a Superdex 200 5/150 column. (C) Circular dichroism spectra of 3 μM IpdAB (blue) or variants E105AA (green), E105AD (black), E58BA (red), R126BM (purple), and R92BM (light blue) in 10 mM sodium phosphate, pH 8.0.  Table 5.2 Specific activity of IpdAB variants IpdAB variant Specific Activity (s-1) WT 1.6 (0.4) E58BA 1.3 (0.3) E105AA <0.01 E105AD <0.01 R126BM <0.01 R92BM <0.01  135  5.3.8 The IpdAB reaction mechanism does not appear to involve a glutamyl-CoA intermediate  Figure 5.6 IpdAB is not inhibited by sodium borohydride. IpdAB was incubated in 50 μl 10 mM sodium phosphate at pH 8.0 for 20 min in the presence of the listed compounds then desalted. (A) Shown are the mass spectra acquired from intact-protein LC-MS on each treated IpdAB sample. Red squares indicate mass of IpdA. (B) The relative specific activity of each NaBH4 treated IpdAB is shown towards 50 μM COCHEA-CoA in the presence of 150 μM CoASH, and 4 μM FadA6 in 200 μl HEPES, pH 7.5 (I= 0.01M). Bars indicate standard deviation (N=3).   In Class I CoTs the glutamyl-CoA intermediate has been trapped by incubating the reaction mixture with sodium borohydride (NaBH4). This reduces the glutamyl-CoA intermediate to a thiohemiacetal which may be observed using LC-MS [227]. This also leads to the enzyme’s irreversible inhibition. To test for the occurrence of a glutamyl-CoA intermediate in the turnover of IpdAB, we incubated a mixture of IpdABRHA1 and COCHEA-CoA with NaBH4. LC-MS analysis of the incubated enzyme revealed no significant difference in mass of either the α (32328 Da) or β (27375 Da) subunits with respect to untreated IpdAB (Figure 5.6A). Moreover, treatment with NaBH4, either in the presence or absence of COCHEA-CoA, did not 136  significantly reduce the specific activity of IpdAB following removal of remaining sodium borohydride (Figure 5.6B). These results strongly indicate that the IpdAB mechanism does not involve a glutamyl-CoA thioester intermediate. 5.3.9 Formation of a β-keto enolate in the E105AA variant The observation of the lactonized COCHEA-CoA in the IpdAB E105AA active site prompted us to test whether binding to IpdAB variants in solution perturbed the absorption spectrum of COCHEA-CoA. Titration of the E105AA variant with COCHEA-CoA yielded a stable yellow colored species, ESyellow (λmax = 312 nm; Figure 5.7 inset) consistent with an enolate. A dissociation constant (KD) for COCHEA-CoA of 0.4 ± 0.2 μM was calculated from the titration data. This species was not observed in WT IpdABRHA1 nor either the Arg92B or Arg126B variantsIpdAB catalyzed deuteration of COCHEA-CoA   Figure 5.7 IpdAB E105A stabilizes a yellow coloured species. Shown is the increase in absorbance at 310 nm upon titration of COCHEA-CoA into 3.5 μM IpdABRHA1 E105AA. The curve represents the best fit of the quadratic binding equation. Insert displays spectra recorded at 0 μM (grey) up to 5 μM (purple) COCHEA-CoA.    137  5.3.10 IpdAB catalyzed deuteration of COCHEA-CoA Although IpdABRHA1 did not detectably transform COCHEA-CoA ([M+H]+ = 976.196 Da) in the absence of FadA6, the observation of ESyellow in the E105AA variant suggested that the enzyme might catalyze a reaction that is not detected using LC-MS, such as the rapid interchange between COCHEA-CoA and the ring-opened MeDODA (Figure 5.2F). We hypothesized that such a reaction would be detected by following deuterium incorporation into COCHEA-CoA from deuterium oxide (D2O). Indeed, incubation of IpdAB and COCHEA-CoA in the presence of D2O resulted in the formation of a new species with [M+H]+ = 979.216 Da (Figure 5.9B), consistent with deuteration at three positions. This species had the same HPLC retention time as COCHEA-CoA, consistent with the two compounds being structurally identical (data not shown). COCHEA-CoA was not deuterated in the absence of IpdAB (Figure 5.9B), nor was the incorporated deuterium exchanged out when the 979 species was incubated in water (data not shown). The activity appears to be specific to COCHEA-CoA as IpdABRHA1 did not catalyze the deuteration of 5αOH-HIC-CoA or any other CoA thioester tested. MS3 indicated that all three sites of deuteration were located on the acyl moiety as the characteristic 428 m/z fragment ion generated from CoA was observed in both species (Figure 5.9C). In contrast, a diagnostic 181.0865 m/z fragment ion assigned as resulting from cleavage of the carboxy thioester in COCHEA-CoA was shifted to 184.1014 m/z in 2[H]-COCHEA-CoA (Figure 5.9C). Incubation of IpdABRHA1 and FadA6 with COCHEA-CoA and CoASH in buffered D2O yielded 2[H]3-MOODA-CoA (m/z = 955.2172 Da; Figure 5.9D) and 2[H]3-acetyl-CoA (m/z = 813.1544 Da; Figure 5.9E). IpdABRHA1 E105AA did not catalyze deuteration of COCHEA-CoA (Figure 5.9F). 138   Figure 5.8 IpdAB catalyzes proton exchange on COCHEA-CoA (A) Diagram depicting predicted fragments from MS3 on COCHEA-CoA. 100 μM COCHEA-CoA was incubated with (red) or without (blue) 2.5 μM IpdABRHA1 in D2O. Shown are the respective mass spectra for the LC-MS/MS of COCHEA-CoA. (B); LC-MS3 (150-275 m/z) of COCHEA-CoA (C); or LC-MS/MS of MOODA-CoA (D) and acetyl-CoA (E) when 5 and 150 μM of FadA6 and CoASH, respectively were added . (E) LC-MS/MS mass spectra of COCHEA-CoA following incubation with IpdABRHA1 WT (red) or E105AA (green) in D2O.   To determine the rate and location of the IpdAB-catalyzed deuteration, the reaction was followed using NMR. A time-dependent decrease in the integration of the C-11 methyl doublet of COCHEA-CoA was observed (Figure 5.10A) indicating exchange at C-5. This loss was not observed in the absence of IpdAB. The initial rate of deuteration at C-5 was calculated using Equation 3 (Figure 5.10B) at 40-50 s-1, although the fit to data beyond 20 minutes was poor, likely due to the competition of 2[H]-COCHEA-CoA for IpdAB. Monitoring the integration of a singlet from a methyl group in the CoA moiety confirmed that COCHEA-CoA did not 139  significantly degrade during this experiment (Figure 5.10, grey). A concomitant decrease in the C-5 proton signal at 2.78 ppm was observed yielding highly similar results when used for measuring the rate of exchange (data not shown). A time-dependent decrease in proton integration in the 2.4-2.6 ppm range was also observed, consistent with deuteration at C-3. However, LC-MS analysis of the COCHEA-CoA pre- and post-incubation with IpdABRHA1 indicated that M + 1 2[H] was the most abundant species, suggesting that the rate of exchange at C-3 is significantly slower than at C-5 (data not shown).  Figure 5.9 NMR characterization of IpdAB catalyzed deuterium incorporation (A) 1H-NMR spectrum displaying the time dependent decrease in the C-10 methyl doublet of COCHEA-CoA upon incubation of IpdABRHA1 in D2O. Blue, red, green, purple, yellow and orange curves depicts before addition of IpdABRHA1 and 6, 11, 21, and 52 minutes after addition, respectively. (B) Time course displaying the relative decrease in proton integration for the C-10 methyl doublet (1.28 ppm; black dots) and the C-36 methyl group (0.75 ppm; grey dots) in the CoA moiety of COCHEA-CoA following addition of IpdABRHA1. Solid curve displays the best fit of Eq. 2 to the initial 20 min of data. Dotted line indicates relative intensity = 1.0 (C) Location of deuteration (D) on COCHEA-CoA as determined by NMR.        140  5.3.11 18O is not incorporated into COCHEA-CoA or IpdAB  Figure 5.10 Additional NaBH4 and 18O experimental data (A) Diagram of proposed IpdAB reaction mechanisms (I and II). Red atom indicates proposed location of 18O from H218O.18O incorporation into COCHEA-CoA. LC- MS mass spectra of 100 μM COCHEA-CoA following incubation with 5 μM IpdAB (B) or (C) 2 μM IpdAB, 20 μM FadA6, and 125 μM CoASH in 10 mM sodium phosphate pH 8.0 prepared in H2O or 97% (18O abundance) H218O. Red box indicates mass spectra of MOODA-CoA.  To test whether the lactonized species observed in the IpdAB·COCHEA-CoA complexes is catalytically relevant, we incubated IpdABRHA1 and COCHEA-COA in the presence of >90% H218O and assayed for 18O incorporation using LC-MS. More specifically, a retro-Claisen like ring-opening reaction could be facilitated by hydrolysis of the lactone (Reaction I, Figure 5.11A), hydrolysis of a glutamyl ester (Reaction II, Figure 5.11A), or direct hydroxylation at C-4. Hydrolysis of the lactone in Reaction I should result in 18O incorporation at C-1 in the presence of H218O. We did not detect any 18O exchange into COCHEA-CoA using LC-MS (Figure 5.11B). Moreover, intact protein LC-MS indicated that 18O was not detectably 141  incorporated into IpdAB (data not shown) as would be expected for the hydrolysis of the resulting acyl-enzyme ester linkage. Considering that carboxylates are not readily exchangeable [228, 229], these results indicated that neither the lactonized COCHEA-CoA observed in the crystal structures nor a Glu105A–ester are catalytically relevant. As a positive control, incubation of IpdAB, FadA6, COCHEA-CoA, and CoASH produced MOODA-CoA with a mass of 954.2 Da, consistent with the incorporation of 18O at C-4 (Figure 5.11C). Although 18O incorporation into MOODA-CoA was observed (Figure 5.11C), 16O - 18O exchange occurred rapidly upon dilution into H2O + 0.1% formic acid prior to LC-MS. Therefore, we were unable to conclude whether 18O incorporation occurred from the hydrolysis of COCHEA-CoA or oxygen exchange at the C-4 ketone of MOODA-CoA after its production [230].   142   Discussion Chapter 6:This thesis characterizes aspects of the catabolism of the latter part of the cholesterol molecule by Mtb. This work provides insights into cholesterol catabolism in related bacteria and, more generally, microbial steroid catabolism. These studies: (A) establish the order of cholesterol side chain and Rings A/B degradation in Actinobacteria (Chapter 2); (B) provide a structural and biochemical model for the HIP-CoA mediated regulation of the KstR2 regulon, present in steroid-degrading Actinobacteria (Chapter 3); (C) elucidate the catabolic steps involved in steroid Rings C and D opening in all known steroid-degrading bacteria (Chapter 4); and (D) characterize IpdAB, a virulence factor with a novel catalytic function for a member of the CoA Transferase superfamily (Chapter 5). More generally, each chapter demonstrates how CoA thioesters play a vital role in cholesterol catabolism and how improved methods to observe CoA thioesters could advance analyses of bacterial catabolic pathways.  6.1 Cholesterol Rings A and B are degraded prior to the alkyl side chain The data presented in Chapter 2 establish that in the catabolism of cholesterol by Mtb and other Actinobacteria, complete Rings A/B degradation precedes that of the alkyl side chain. Disruption of the genes encoding the last two Rings A/B-degrading enzymes, hsaC and hsaD, in RHA1 led to the accumulation of metabolites with incompletely degraded side chains when the strains were incubated with cholesterol. Similarly, both HsaDMtb and HsaDMab had higher specificity (kcat/KM values) for substrates with partially degraded side chains. These data extend the findings of Capyk et al. (2011) and Casabon et al. (2013) in which the alkyl side chain was shown to be degraded somewhere after the KshAB and before the FadD3 reactions, respectively, requiring updates to the cholesterol catabolic pathway [69, 79].  143  6.1.1 Steroid degradation pathways in Mab appear to share a single HsaD Mab grew on both cholesterol and 4-AD, consistent with containing gene clusters responsible for the catabolism of each steroid. Growth on cholesterol was very similar to what has been reported in Mtb and other Actinobacteria [51, 79, 187]. However, Mab grew to much lower yields on 4-AD, in contrast to what was reported in M. smegmatis [27]. Moreover, although the two strains contain similar 4-AD catabolic genes, apparently under control of a PadR-family transcriptional repressor, growth of M. smegmatis on 4-AD was only observed in strains lacking either this repressor or the KstR regulator [27]. The effector of the PadR repressor remains unknown: the 4-AD catabolic genes appear to be constitutively expressed in M. smegmatis and were not up-regulated in the presence of either 4-AD or cholesterol [27]. Moreover, the mechanism of uptake of 4-AD by M. smegmatis and Mab is unclear as actinobacterial Mce4 transporters are specific to steroids with an alkyl side chain [54]. Interestingly, despite the absence of dedicated 4-AD catabolic genes, Mtb also appears to grow on this steroid [51]. A unique feature of the cholesterol and 4-AD catabolic pathways of Mab is that they likely share a single HsaD homolog. All other steroid catabolic clusters characterized to date in Actinobacteria have a complete set of Rings A/B-degrading enzymes [22, 25, 27]. However, the steady-state kinetic data establish that HsaDMab transforms both DSHBNC-CoA and DSHA, respectively, with kcat/KM values similar to those of other MCP hydrolases for their respective physiologically substrates [156, 189]. It is unclear whether the substrate specificity of HsaDMab reflects that of all Subfamily V MCP hydrolases.  The data do not exclude the possibility that Mab_4366c catalyzes the hydrolysis of steroid-derived MCPs in M. abscessus. Although Mab_4366c occurs within a cluster of genes 144  predicted to encode the catabolism of isopropyl benzene, its phylogeny differs from characterized IpbDs/CumDs (Figure 2.1) [191, 192]. The unique phylogeny of Mab_4366c appears to relate to two regions that share low amino acid identity with characterized MCP hydrolases, Pro76-Val91 and Ala136-Pro163. Based on modeling studies, the latter contributes to the “non-conserved loop” and “non-polar” (NP) subsite in MCP hydrolases [162]. The NP subsite is a major determinant of substrate specificity. This suggests that Mab_4366c may have divergently evolved from a Subfamily IV ancestor to accommodate non-steroidal substrates. Steady-state kinetic analyses of Mab_4366c with DHSA, DSHBNC-CoA and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate would help evaluate the enzyme’s role in steroid catabolism. Similarly, deletion mutants of hsaDMab and mab_4366c would be invaluable in validating their respective roles.  6.1.2 HsaDMtb and HsaDMab have highest specificity for steroid substrates with incompletely degraded side chains The specificity of HsaDMtb for DSHBNC-CoA over DSHA (kcat/KM values) is greater than in HsaDMab. This is consistent with what has been reported for other cholesterol Rings A/B-degrading enzymes, such as KshA from Mtb and KshA1 from Rhodococcus rhodochrous [41, 69] as well as the extremely low substrate specificity of HsaAB for a substrate with a completely degraded side chain [76]. Interestingly, in the crystal structure of HsaDMtb·DSHA (PDB# 2WUF), the 17 oxo of DSHA is orientated towards an atypically large, polar pocket at the entrance to the enzyme’s active site [77]. Moreover, the constellation of charged and polar residues in this pocket is reminiscent of what has been reported in other CoA-binding enzymes [231]. Residues within this polar pocket are generally conserved across actinobacterial HsaDs, however not in HsaDMab. Unfortunately, attempts to obtain a structure of HsaDMtb·DSHBNC-145  CoA were unsuccessful (data not shown). Preliminary studies using molecular docking of adenine into this pocket implicated Ile144, Asp150, and Trp223 as being significant in enzyme-substrate binding (data not shown). Site-directed mutagenesis of these residues in the HsaDMtb S114A variant displayed 5-10 nm bathochromic shifts in ESred when titrated with DSHBNC-CoA suggesting perturbation of substrate binding. However, these mutations also destabilized the enzyme’s structure making the titration data difficult to interpret (data not shown). Overall, the architecture of HsaDMtb is consistent with it accommodating a CoA sidechain substrate, however identification of the exact determinants of CoA binding require additional data. 146  6.1.3 Updates to the pathway of cholesterol degradation  Figure 6.1 Proposed pathway of cholesterol catabolism in actinobacteria.  Enzymes for which in vitro activity exists with the substrate depicted are shown in bold. Substrates of Rings A/B catabolic enzymes shown with isopropionyl-CoA moiety. However, the isopentanoyl-CoA or isooctanoyl-CoA analogs cannot be excluded as physiological substrates.   An update to the pathway for cholesterol degradation is proposed (Figure 6.1). Following cholesterol uptake [33], C-3 dehydrogenation [72, 232], C-27 oxidation [58], and CoA thioesterification at C-27 [179] the resulting, 3-oxo-4-cholestenoyl-CoA is degraded via two rounds of β-oxidation involving ChsE4-ChsE5, ChsE3, and FadA5, yielding a propionyl-CoA and an acetyl-CoA molecule [47, 63, 67, 233]. Prior to removal of the C-17 isopropionyl-CoA 147  moiety, KstD forms 1,4-BNC-CoA and cholesterol Ring A is aromatized by KshAB yielding 3-hydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,5(10)-trien-22-oyl-CoA (HSBNC-CoA) [69]. HsaAB presumably hydroxylates HSBNC-CoA to DHSBNC-CoA which undergoes meta-cleavage by HsaC forming DSHBNC-CoA [76]. DSHBNC-CoA is cleaved into HHD and 3aα-H-4α(3’-propanoate)-7aβ-methylhexahydro-5-indanone-1-propionyl-CoA (HIDP-CoA) where side-chain degradation continues likely involving ChsE1-ChsE2 and ChsH1-ChsH2 [63, 67]. Finally, a currently unidentified thiolase or aldolase removes the final propionyl-CoA moiety generating HIP which feeds into Rings C and D degradation. Consistent with the updated pathway, deletion of the intracellular growth (igr) operon in Mtb, containing chsE1, chsE2, chsH1, chsH2 and ltp2, yields extracellular HIDP accumulation in the presence of cholesterol [48, 66]. Furthermore, HSBNC accumulates when Mtb is treated with an HsaA inhibitor in the presence of cholesterol [134].  6.2  HIP-CoA binding to KstR2 regulates catabolism of cholesterol Rings C and D Chapter 3 provides the first molecular insights into the KstR2-mediated regulation of the expression of steroid catabolic genes in Mtb and other actinobacteria. The structural and titration data establish that the KstR2 dimer binds 2 molecules of HIP-CoA. Each HIP-CoA molecule binds in a deep cleft that spans the KstR2 dimer with the adenosine and HIP moieties bound by separate protomers. The extensive electrostatic and hydrophobic interactions that mediate the binding of HIP-CoA to KstR2Mtb are corroborated by the high affinity of the regulator for its effector molecule. The functional significance of the KstR2Mtb·HIP-CoA structure was further validated by directed mutagenesis, which established that Arg162 and Trp166 contribute significantly to the binding of HIP-CoA while minimally affecting the binding of the operator DNA. Finally, comparison of the KstR2Mtb·HIP-CoA, KstR2RHA1 and SlmA·DNA structures 148  suggests how effector binding alters the conformation of the regulator to relieve binding of the operator DNA. 6.2.1 Comparisons to TetR family of transcriptional repressors The amino acid sequence conservation among KstR2 orthologs in steroid-degrading Actino-bacteria further validates the functional importance of the residues identified in the KstR2Mtb·HIP-CoA structure. These orthologs share ~50% amino acid sequence identity with higher conservation among DBD residues predicted to bind the operator DNA and EBD residues that bind HIP-CoA. Specifically, the residues located on helices α2 and 3 in KstR2Mtb are all conserved with the exception of Gly39. This is consistent with the conserved nucleotide sequence of the operator across Actinobacteria [83]. Similarly, 19 of 23 residues that contact the effector in the KstR2Mtb·HIP-CoA complex, located between residues 138 to 195, are conserved in KstR2 orthologs. Comparison of the structures of FadRs from Thermus thermophilus (3ANP, 3ANG; [88]) and Bacillus subtilis (1VI0; [234]) in complex with fatty acyl-CoA are strikingly similar to the KstR2Mtb·HIP-CoA complex: in each, the ligand is bound in a cleft that spans the two protomers with the fatty acid and adenosine moieties bound to separate chains. Conserved residues include Arg159 and Arg173 (KstR2 numbering) that interact with the adenine and Arg162’ which hydrogen bonds with the diphosphate moiety of CoA. Nevertheless, the adenine ring in the FadR·fatty acyl-CoA complexes is flipped 180o with respect to that in the KstR2Mtb·HIP-CoA complex and oriented perpendicular to the dimer’s rotational axis. By contrast, the fatty acyl-CoA binds in a different way in the DesT complexes, with the CoA moiety at the top of the EBD and the acyl chain extending down a channel, parallel to the α-helices of the EBD [235]. Consistent with the different binding mode, none of the Arg residues are conserved in DesT. The 149  three conserved Arg residues are also not conserved in TFRs that bind smaller effectors, such as QacR of Staphylococcus aureus (PDB 1JT6), Pseudomonas putida TtgR (PDB 2UXI), and Streptomyces coelicolor ActR (PDB 3B6A). Conservation of the CoA-binding residues in the KstR2 and FadR may be extended to other TFRs to gain insight into their respective effectors. Comparison of sequence topology maps of 48 structures of TFRs identified in Yu et al. (2010) indicate that 13 contain at least two of the three conserved basic residues in orientations permissive to CoA binding [165]. Of note, Fad35R of M. tuberculosis (PDB 4G12) regulates the expression of fad35, which encodes an acetyl-CoA synthetase involved in fatty acid degradation [236]. Although Fad35R binds tetracycline, other evidence suggested that a fatty acyl-CoA could be the physiological effector [236]. The presence of Lys184 on the α8-α9 loop, Arg166 on the apical end of helix α8, and His-170 in the middle of helix α8 supports this hypothesis. T. thermophilus HB8 PfmR (PDB 3VPR), which is predicted to regulate PAA or fatty acid catabolism possess basic residues on the α8-α9 loop and middle of helix α8 (Arg163 and Arg149, respectively) but lacks a basic residue at the top of helix α8. Although the respective effectors of these 13 TFRs have yet to be identified, it appears that a significant subset of TFRs bind CoA thioesters. The proposed mechanism of response of KstR2Mtb to HIP-CoA is similar to what has been proposed for other TFRs where the binding of a small molecule effector induces conformational changes that abrogate the regulator’s interactions with its operator DNA [164, 165]. In the absence of a KstR2Mtb·DNA structure, the comparative analysis between the KstR2Mtb·HIP-CoA and SlmA·DNA complexes unveiled differences in the relative conformation of DBD domains in the TFRs that, in case of KstR2Mtb, are likely triggered by HIP-CoA binding. More specifically, HIP-CoA binding to KstR2Mtb repositions helices α4 and α6 causing a net 15o outward rotation 150  of DBD helix α1, displacing helices α2 and α3 compared to corresponding elements in SlmA·DNA complex (Figure 3.4). Interestingly, Tyr48 and His50 on DBD helix α3 in KstR2Mtb·HIP-CoA are rotated outward by ~10 Å as compared to the ligand-free structure of KstR2RHA1 (Figure 3.3). These residues are highly conserved in TFRs and, in E. coli TetR, directly interact with the bases in the major groove of the DNA [165]. Lastly, Lys54, which is also highly conserved in TFRs [165], is displaced by ~7 Å in KstR2Mtb·HIP-CoA compared to KstR2RHA1. The effect of HIP-CoA on KstR2 is also remarkably similar to the displacement of helix α4 and outward rotation of helix α3 observed in apo- vs. ligand bound B. subtilis FadR [87]. In TetR, effector binding thermodynamically stabilizes the DBD in a conformation that is incompatible with DNA binding, preventing the DBD from assuming a conformation that is competent for DNA binding [237]. The inability of KstR2Mtb·HIP-CoA to bind DNA likely has the same mechanistic origin.    The binding of two KstR2Mtb dimers to its operator was somewhat unexpected considering that the KstR box of 14 bp [83] is too short to accommodate four recognition helices in the major groove. Typically the TFR operators that bind two dimers are at least 22 bp in length [205, 238-240]. By contrast, TFRs that bind operator sequences of less than 17 bp bind as a single dimer [239]. It is possible that the KstR2 box extends beyond the 14 bp identified by Kendall et al. [83]. Alternatively, the KstR2 dimers may bind opposite sides of the DNA helix, as in the SlmA·DNA complex (PDB: 4CGT). In such a scenario, the first KstR dimer induces a conformational change in the DNA that facilitates the binding of the second dimer to a non-canonical sequence. Intriguingly, KstR2Mtb contains the Arg-X-Thr motif that is present in SlmA and induces a kink in the DNA [205]. Similar models have been invoked to explain cooperative binding in each of two other TFRs, QacR and CprB [239, 240], although QacR lacks the Arg 151  residue and does not induce a kink in the DNA. Interestingly, single KstR2 boxes occur between divergently transcribed promoters [83] and gel shift assays indicate that KstR2 binds to each of the three boxes with the same stoichiometry [91]. The binding of two KstR2 dimers to opposite sides of the DNA helix may enable the repressor to act at both promoters. Additional experiments and structural data are required to determine the precise architecture of the KstR2Mtb·operator complexes and how this regulates divergently transcribed promoters. 6.2.2 Insights into the inducer of KstR The KstR2∙HIP-CoA model predicted that the effector of M. tuberculosis KstR (PDB# 3MNL) is also a CoA thioester. KstR2Mtb and KstRMtb share only 18% amino acid sequence identity. However, the conserved residues include four that mediate CoA binding in KstR2 (Arg162, Asp163, Trp171 and Arg173) and two that mediate steroid binding (Trp166 and Phe70). Since the publication of Chapter 3, 3-oxo-cholest-4-en-26-oyl-CoA was confirmed to be the effector of KstRMtb in collaboration with the Lott Lab at the University of Auckland [86]. Therefore, the effector of KstRMtb mirrors the regulatory logic of the KstR2 regulon in M. tuberculosis in that the catabolic genes are induced by the first CoA thioester catabolite produced. More particularly, C-27 of cholesterol’s alkyl side chain is oxidized to a carboxylate by either Cyp125 or 142 [58], and thioesterified by FadD19 [179]. Subsequent structural and biochemical analysis demonstrated a mechanism of gene repression analogous to that described for KstR2 by HIP-CoA. Overall, the identification of the effector of KstR demonstrates how structural features of KstR2Mtb∙HIP-CoA can be applied to guide the identification of physiological effectors for other TetR family transcriptional repressors. 152  6.3 Elucidation of the catabolism of cholesterol Rings C and D Until this work, the pathway by which the last two steroid rings, contained in HIP, are degraded in bacteria was unknown. By profiling the culture supernatants and CoA metabolomes of deletion mutants of HIP catabolic genes, ipdAB, ipdC, ipdF, echA20, and fadE32 in Mtb, M. smegmatis, and/or RHA1, three characteristic HIP catabolites were identified: 5αOH-HIC-CoA, COCHEA-CoA, and MOODA. Enzymological experiments further identified HIEC-CoA and MOODA-CoA. When viewed together, the mutant data, enzymological transformations, and bioinformatics analyses, elaborate a pathway for HIP catabolism. 6.3.1 The catabolic pathway of cholesterol Rings C and D in actinobacteria  A model for HIP degradation was proposed in which Ring D cleavage precedes that of Ring C (Figure 6.2). In the proposed pathway, the isopropionyl side chain of HIP is first degraded via β-oxidation to yield 5-OH HIC-CoA. This is then transformed to HIEC-CoA by IpdF and IpdC, before undergoing two successive ring-cleavage reactions: EchA20-catalyzed hydrolysis of Ring D followed by IpdAB-catalyzed hydrolysis of Ring C. Thiolysis of the Ring C-opened product, potentially by FadA6 or another thiolase, yields MOODA-CoA which is then oxidized to 2Δ-MOODA-CoA by an ACAD comprised in whole or in part by FadE32. Although the fate of 2Δ-MOODA-CoA is unclear, a final round of β-oxidation to yield 2-methyl-β-ketoadipyl-CoA (MβKA-CoA) is proposed. This could then be cleaved to propionyl-CoA and succinyl-CoA in a manner analogous to the cleavage of β-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA in the final step of the β-ketoadipate pathway used in the bacterial catabolism of aromatic compounds [241]. While several aspects of the HIP pathway have yet to be elucidated, the data support the proposed physiological roles of four enzymes: IpdF, IpdC, EchA20, and IpdAB.  153   Figure 6.2 Proposed HIP catabolic pathway. NMR-confirmed metabolites are in blue. Metabolites for which MS data were obtained are in black. Other metabolites are in grey. *The current study established that IpdF has this activity, but its physiological relevance is unclear. **Role of FadE30 assigned previously [23] The proposed pathway provides an important framework to further characterize various aspects of steroid metabolism, including the identity of specific metabolites, such as HIEC-CoA and MβKA-CoA, as well as enzymatic steps, such as those catalyzed by Rv3548c and Rv3549c, encoded by the KstR2 regulon. The model further suggests the identities of the fadE-encoded ACADs that act on HIP-CoA and MOODA-CoA, respectively. Another unknown aspect of the pathway is the significance of the IpdF-catalyzed reaction: it is unclear why the 5-oxo group would be reduced and then reoxidized. Anecdotally, chemical synthesis of HIC-CoA proved difficult due to the propensity of HIC-CoA to undergo acid catalyzed ring opening whereas 5OH-HIC-CoA was stable (Hurst, unpublished). Finally, this pathway also predicts that cholesterol feeds into central metabolism via four propionyl-CoAs, four acetyl-CoAs, one 154  pyruvate and one succinyl-CoA. Notably, propionyl-CoA, a potentially toxic metabolite [49], is derived from all three parts of cholesterol: the side chain, Rings A/B and Rings C/D. 6.4 IpdAB is a ring-cleaving hydrolase Among the enzymes whose functions were assigned, that of IpdAB was unexpected based on the bioinformatic analyses (Table 6.2). More specifically, no Class I CoA transferase has been reported to catalyze a retro-aldol hydrolysis. This study provides evidence that IpdAB transforms COCHEA-CoA, thereby catalyzing the hydrolytic opening of the last ring in the bacterial catabolism of steroids. The data further indicate that this reaction does not involve CoA transfer despite IpdAB’s striking similarity to Class I CoTs. First, IpdABRHA1 did not catalyze the transfer of CoA from small acyl-CoA donors to small acids as is typical for Class I CoT. Second, the IpdAB active site lacks key elements that are conserved in CoTs including the catalytically essential glutamate and an oxyanion hole to accommodate the CoA thioester [175]. Third, NaBH4 did not detectably inhibit IpdAB, nor did it trap a glutamyl-CoA thioester intermediate. Finally, the position of the CoA in MOODA-CoA, the product of the IpdABRHA1/FadA6 reaction, indicates that the CoA isn’t transferred from one carbon to another prior to thiolysis. Therefore, the data support IpdAB as transforming COCHEA-CoA into 6-methyl-3,7-dioxodecanedioyl-CoA (MeDODA-CoA).  155  6.4.1 Proposed mechanism of IpdAB  Figure 6.3 Proposed mechanism of IpdAB. Location of O atom from H2O is identified in red.  Based on the presented data, a mechanism for IpdAB involving a retro-Claisen like hydrolysis (Figure 6.3) is proposed. In this mechanism, binding of COCHEA-CoA to the IpdAB active site stabilizes the C-8 enolate resonant form of COCHEA-CoA via hydrogen bonding from Arg92B and Asn131B. A solvent species, activated by either Glu105A or the C-1 carboxylate, attacks at C-4, which is more electrophilic due to stabilization of the enolate. Tautomerization of the C-8 enolate protonates C-9. Deprotonation of the resulting C-4 hydroxyl yields an alkoxide anion, whose ketonization and concerted protonation at C-9 permits the cleavage of the C-4/C-9 bond and Ring C opening. The proposed mechanism is supported by several lines of evidence. First, the E105AA variant stabilizes a species whose spectrum is consistent with a CoA-enolate, as exemplified by the acetoacetyl-CoA-Mg2+ complex (λmax = 310 nm [242]). The structure of the IpdAB·COCHEA-CoA complex suggests that Arg92B stabilizes the C-8 enolate. Second, deuteration at C-5 and C-3 is consistent with the increased electrophilicity of C-4. The failure of the E105AA variant to catalyze deuteration of COCHEA-CoA indicates that deuteration occurs 156  concerted with or after hydroxylation at C-4. Further, the rate of deuteration is an order of magnitude faster than COCHEA-CoA turnover and is thus not rate-limiting. Finally, the lack of 18O incorporation at C-1 indicates that the lactonized COCHEA-CoA is not catalytically relevant although the nucleophile could be activated by the C-1 carboxylate. Several aspects of the mechanism remain to be elucidated. First, although the proposed mechanism posits Glu105A as the base that activates the nucleophile, the structural data indicate that both this residue and the C-1 carboxylate may be positioned to activate water and/or deprotonate the C-4 hydroxyl. Nevertheless, it is unclear whether the C-1 carboxylate is catalytically essential. Additional mechanistic studies involving the substrate were complicated due to limited quantities of COCHEA-CoA and the inability to directly observe its hydrolysis product despite various attempts to chemically trap MeDODA-CoA (data not shown). It is possible that MeDODA-CoA was not observed because the equilibrium favours COCHEA-CoA and thiolysis of MeDODA-CoA drives the hydrolytic reaction forward. Indeed, the possibility that IpdAB catalyzes an intramolecular CoA transfer from C-11 of COCHEA-CoA to C-11 in MeDODA-CoA cannot be excluded. However, this is not supported by the results of the CoA transferase studies and is thermodynamically unfavourable as formation of a carboxylic acid or ester would increase the pKa of hydrogens at C-9, increasing the activation energy for C-4/C-9 hydrolysis. 6.5 Broader implications from the elucidation of HIP catabolism 6.5.1 The HIP catabolic pathway in other bacteria The occurrence of a single KstR2 regulon in Actinobacteria in strains that contain several distinct steroid catabolic pathways [22, 25] suggests that hydroxylated HIP-CoA can act as the effector of at least some KstR2 orthologs. For example, RHA1 possesses at least three distinct 157  pathways that converge on the HIP catabolic pathway, two of which are responsible for cholesterol and bile acid catabolism, respectively [22]. The catabolism of bile acids such as cholate results in the production of 3’OH HIP [79], suggesting that 3’OH HIP-CoA would be an effector of KstR2RHA1. Inspection of the KstR2Mtb·HIP-CoA structure indicates that there is sufficient space adjacent to C-3’ of the HIP moiety to accommodate a hydroxyl group. Moreover, all of the residues within a radius of 6 Å of C3’ are conserved in KstR2RHA1 (Figure 3.3). By contrast, the binding of 7β-OH HIP-CoA is predicted to be sterically hindered by Tyr-108 (Tyr-112 in KstR2RHA1). This is consistent with the finding that C-12 hydroxyl groups of bile acids (corresponding to C-7 of HIP) are removed prior to Rings C/D degradation in Actinobacteria [28, 79]. By contrast, 7β-OH HIP is produced in steroid-degrading Gram negative bacteria such as Pseudomonas putida DOC21 [21]. However, the HIP catabolic genes in these strains appear to be regulated by a LuxR-type transcriptional repressor [18, 21]. The HIP catabolic genes are conserved in steroid-degrading bacteria for which genome sequence data are available, suggesting that the pathway is not only employed in the degradation of steroids other than cholesterol [18, 22, 25], but also in the anaerobic degradation of steroids [20]. Indeed, differences in the HIP catabolic gene cluster in diverse bacteria appear to reflect the different steroid-catabolizing capabilities of the strains. For example, in bacteria that catabolize cholate or other bile acids, the HIP catabolic gene cluster contains echA13 (RHA1_RS22405 in RHA1) [17, 22, 91]. A homolog of EchA20, EchA13 is proposed to remove the hydroxyl of 7β-OH HIP, generated from cholate degradation [21, 91], and is not present in Mtb, which does not degrade cholate. Similarly, the HIP catabolic gene cluster of S. denitrificans DSM 18526, which is up-regulated during the aerobic and anaerobic catabolism of testosterone [20], lacks a homolog 158  of fadD3/stdA3. Instead, this strain contains a homolog elsewhere in the genome: ACG33_09100 shares 53% amino acid sequence identity with StdA3 of P. putida DOC21 [21]. The genomic context of fadD3 in S. denitrificans DSM 18526 may reflect the possibility that the β-oxidation of steroid Rings A and B yields HIP-CoA directly, obviating the need for FadD3 in anaerobic steroid catabolism.  6.5.2 Cleavage of MeDODA-CoA likely requires an unidentified thiolase The low turnover rate of FadA6 in the coupled reactions indicates that it may not be the physiological thiolase responsible for MOODA-CoA formation. Indeed, FadA5Mtb, which acts on the alkyl side chain of cholesterol [47], turned over MeDODA-CoA at the same rate as FadA6 in the coupled reaction (data not shown). FadA6 may be required in the cleavage of 3’oxo- 5-OH-HIP-CoA to 5αOH-HIC-CoA, upstream of COCHEA-CoA in the HIP catabolic pathway, and the enzyme acting on MeDODA-CoA may be encoded outside of the KstR2 regulon (Figure 6.2).  6.5.3 HIP catabolism displays similarities with anaerobic aromatic catabolism A reoccurring theme in Rings C/D degradation is the similarities with bacterial aromatic catabolism. The hydrolytic cleavage of the cyclohexenone ring by IpdAB is similar to that catalyzed by Oah, a crotonase involved in the anaerobic degradation of benzoate in Thauera aromatic [243]. Oah hydrolyzes the cyclohexene ring of 6-oxocyclohex-1-ene-1 carbonyl-CoA, a structural analog of COCHEA-CoA, by hydroxylating a double bond. The ensuing retro-Claisen opening of the cyclohexanone ring is comparable to that of IpdAB (Figure 6.3) however, carbon-carbon cleavage occurs adjacent to the carbon double bond yielding 3-oxopimeloyl-CoA, a β-keto CoA thioester analogous to MeDODA-CoA [243]. Interestingly, the subsequent catabolism of 3-oxopimeloyl-CoA and MOODA-CoA are predicted to be similar [178, 244].  159  The phylogeny of ipdAB implies divergent evolution from ancestral genes encoding a Class I β-keto-CoA transferase to facilitate the opening of steroid Ring C. Interestingly, the degradation of HIP is predicted to yield methyl-β-ketoadipyl CoA (Figure 6.2). This metabolite is similar to β-ketoadipyl-CoA, the product of the Class I β-keto- CoA transferase, PcaJI, involved in benzoate degradation [245]. HIP catabolism is not predicted to require a β-ketoadipate CoA transferase like PcaJI. However, the subsequent catabolic step in both pathways involves a thiolase, and generates succinyl-CoA and either acetyl-CoA or propionyl-CoA and [245]. It is possible that duplication of the β-ketoadipate pathway may have facilitated the repurposing and divergent evolution of an ancestral PcaJI homolog to generate IpdAB.  6.6 Insights for Mtb pathogenicity and therapeutic development 6.6.1 Disruption of KstR2 genes yield a ‘cholesterol-dependent-toxicity’ in Mtb Deletion of ipdAB and ipdC in Mtb yielded strains that failed to grow on glycerol in the presence of cholesterol and, in the case of ipdAB, significantly slowed the growth in THP-1 derived macrophages. These strains displayed distinct differences in the concentration and identity of CoA thioesters and CoASH. This suggests that ‘cholesterol-dependent toxicity’ of the mutants may be due to the sequestration of CoASH, making it unavailable for other cellular processes. Consistent with the sequestration hypothesis, strains which grow in the presence of cholesterol (ΔfadD3 RHA1, ΔfadE32 M. smegmatis, and ΔipdF M. smegmatis) failed to accumulate significant levels of unique, cholesterol-derived CoA thioesters [79]. This implies that low intracellular CoASH concentrations in cholesterol catabolic gene mutants in Mtb may be predictive of cholesterol-dependent toxicity. The lack of toxicity in the ΔipdF and ΔfadE32 mutants could reflect the ease of hydrolysis of the corresponding CoA thioesters (e.g., MOODA-160  CoA to MOODA) and/or the presence of compensatory enzymatic activities (e.g., other cellular dehydrogenases could catalyze oxidation of the 5-OH HIC-CoA hydroxyl).  Disruption of cholesterol catabolic genes is reported to generate both attenuated and avirulent strains of Mtb due to the predicted accumulation of toxic cholesterol-derived metabolites [39, 47, 51]. For example, the reduced virulence of ΔhsaC Mtb in guinea pig infection models has been attributed to the accumulation of toxic catechols resulting from DHSA accumulation [39]. However, DHSBNC-CoA accumulation, implied by the data in Chapter 2, suggests CoA sequestration may also contribute to this attenuated phenotype. Interestingly, disruption of the ratio between acetyl-CoA and propionyl-CoA in Mtb during growth on cholesterol has been reported to result in a toxic phenotype [112]. Indeed, reduction in CoASH in strains displaying cholesterol-dependent toxicity typically coincided with reduced acetyl-CoA levels in the CoA metabolic data (data not shown). Although additional data are required to test the CoASH sequestration hypothesis, the data rationalizes the effectiveness of the ipdAB mutant as a vaccine in R. equi and indicates which HIP degradation enzymes would be good targets for future therapeutic development. 6.6.2 Therapeutics targeting Mab The role of steroid catabolism in Mab infection is unclear. Resolving this question is complicated by a scarcity of effective genetic tools and infection models [246]. Mab was recently shown to replicate in bone marrow-derived murine macrophages [247]. However, it has not yet been established whether Mab is an obligate intracellular pathogen. Developing cholesterol catabolism as a target for novel therapeutics in Mab is dependent on the development of reliable infection models. By analogy, inhibition of cholesterol utilization by Mtb was 161  achieved using small molecules targeting HsaAB, HsaD, an adenylate cyclase as well as other undetermined cholesterol catabolic genes [53, 134, 136]. Although it is unclear whether any of these small molecules will be effective towards inhibiting cholesterol catabolism in Mab, the occurrence of CoA thioester metabolites in the degradation of cholesterol Rings A/B increases the value of these enzymes as targets for inhibitors given the proposed toxicity of CoA sequestration [178]. 6.6.3 Therapeutics targeting IpdAB The data presented herein corroborates previous work which identified IpdAB as essential for virulence [23, 52] as ΔipdAB Mtb was attenuated in MΦ and failed to grow on cholesterol in the presence of another carbon source (Chapter 4). The characterization of IpdAB provides insights for the rational design of IpdAB inhibitors. It is conceivable that a non-hydrolyzable cyclopentane ring structure about C-4 of COCHEA-CoA, or any lactonized COCHEA-CoA analog thereof, could effectively bind the active site, inhibiting IpdAB. Similarly, the proposed mechanism requires a carbon-carbon double bond at C-4/C-5 of COCHEA-CoA for its hydrolysis. Presumably, saturation of this bond should not significantly alter binding, but would prevent hydroxylation at C-4.  Currently, no inhibitors of IpdABMtb have been reported in the literature. Unfortunately, the cholesterol-dependent-toxicity observed in ΔipdAB RHA1 is suppressed by the deletion of fadD3 (Figure 4.3). Therefore, spontaneous suppressor mutants could diminish the effectiveness of therapeutics targeting IpdAB. Additionally, FadD3Mtb can be reversibly inhibited via post translational modification (Brown, unpublished) permitting the bacteria to arrest HIP catabolism, alleviating toxicity related to IpdAB inhibition. These processes may explain why Vanderven et al. failed to observe small molecules that target IpdAB or any cholesterol Rings C and D 162  degradation enzyme [134]. Although this diminishes the hope of IpdAB inhibitors as monotherapies, they may still prove effective in combination therapies.  6.7 Remaining questions and future directions 6.7.1 Cholesterol side chain and Rings A and B degradation The identification of concurrent side chain and Rings A and B degradation implies that HsaAB acts on a substrate with a partially degraded side chain. Unfortunately, the specificity of HsaAB for HSBNC-CoA could not be reliably determined (data not shown). This suggests that either HSBNC-CoA is not the physiological substrate of HsaAB or that the assay conditions for HsaAB do not reflect its physiological activity. Due to the identification of multiple inhibitors of HsaAB that are active in vivo [134] and the poor substrate specificity towards 3-HSA [76], additional investigations of HsaAB with HSBNC-CoA are warranted.  Due to the limited availability of isopropionyl-CoA derivatives of steroid metabolites, I was unable to ascertain the precise alkyl-side chain intermediate for which HsaDMtb and HsaDMab have highest specificities. The physiological substrate of HsaD may contain an isopropionyl-, isopentanoyl, or isooctanoyl-CoA moiety with an array of different β-oxidative desaturation or hydroxylations thereof. Interestingly, the large entrance to the active site in HsaDMtb may permit promiscuity towards a wide range side chain intermediates [77]. However, only metabolites with isopropionyl side chains were observed in ΔhsaC and ΔhsaD RHA1, suggesting that substrates with an isopropionyl-CoA side chain may be preferred by HsaD.  6.7.2 Regulation of cholesterol catabolism In addition to the transcriptional regulation of cholesterol and HIP catabolism by KstR and KstR2, respectively, this catabolism is also regulated at the post-translational level. Acyl-CoA synthetases (FadD) catalyze the production of the respective effectors for KstR and KstR2 163  [79, 179]. FadDs are regulated via the reversible acetylation of their catalytic lysine, which inhibits catalytic activity. Acetylation is effected by protein lysine acetyltransferases (PAT) which, in turn, are regulated allosterically by cAMP [248-250]. Deacetylation of FadDs is catalyzed by deacetylases [250]. Recently, a number of small molecules that inhibit growth of Mtb on cholesterol and in MΦ were discovered that target an adenylate cyclase encoded by rv1625c [134, 136], implying a regulatory role for cAMP in cholesterol and/or HIP catabolism. To date, the precise involvement of cAMP is unknown in these pathways, however FadD3 is acetylated by a PAT in Mtb (Mt-PAT), suggesting Mt-PAT and cAMP mediated regulation (K. Brown, unpublished). Furthermore, KstR-regulated genes are up-regulated during overexpression of hypoxia related regulators, Rv0081 and DosR [188]. Although the Mce4 genes are up-regulated in the presence of cholesterol and are predicted to be KstR-regulated [251], overexpression of KstR did not lead to repression of the Mce4 operon [252, 253]. Similarly, chromosome immuno-precipitation (ChIP) of KstR failed to identify a KstR-binding site upstream of the mce4 genes [252]. Overall, the additional regulatory mechanisms of cholesterol and HIP catabolism warrant future investigation. 6.7.3  Elucidation of HIP catabolism The metabolic steps between 5OH-HIP-CoA to 5OH-HIC-CoA and MOODA-CoA to central metabolism were not elucidated (Figure 6.2) in Chapter 4. Presumably, 5αOH-HIP-CoA undergoes β-oxidation to form 5OH-HIC-CoA likely involving the short chain dehydrogenases, Rv3548c and Rv3549c, and/or the acyl-CoA dehydrogenases, FadE30 and FadE33 [25]. Preliminary characterizations of M. smegmatis with respective gene deletions of rv3548c, rv3549c, fadE30, and fadE33 support our current functional assignment (data not shown). In contrast, ΔfadA6 M. smegmatis accumulated metabolites upstream of COCHEA-CoA 164  corroborating its low specific activity towards MeDODA-CoA. Overall, validation of these mutants was hindered by our inability to produce HIPE-CoA (Δ3’-5OH-HIP-CoA, Figure 6.2) for use in in vitro enzyme studies. Future studies should focus on producing FadE30 for the production of this metabolite. 6.7.4 Characterization of IpdAB IpdABs make up a subfamily with a distinct catalytic function within the CoA transferase superfamily. However, it is unclear whether such enzymes are unique to steroid catabolism. Preliminary searches of actinobacterial genomes failed to identify annotated CoA transferases with conserved IpdAB characteristic residues such as Glu105A, Gly57B, Arg126B, or Arg92B. Unfortunately, amino acid sequence conservation with CoA transferases limit the effectiveness of BLAST searches for IpdAB paralogs in bacterial genomes. Given the ubiquity of steroid catabolism in bacteria, and the observation of COCHEA-CoA like catabolites in non-steroidal metabolic pathways [243], the preponderance of evidence would point to there being unidentified members of the IpdAB subfamily in non-steroidal catabolic pathways.  165  Bibliography  1. Makin, H.L.J.a.G., D.B. (2009): Springer. 2. Singh, P. (2016) Frontiers in Cell and Developmental Biology. 4: p. 156. 3. Miras-Moreno, B., Sabater-Jara, A.B., Pedreno, M.A. and Almagro, L. (2016) Journal of Agricultural Food Chemistry. 64(38): p. 7049-58. 4. Goluszko, P. and Nowicki, B. (2005) Infection and Immunity. 73(12): p. 7791-6. 5. Chyu, K.-Y., Lio, W.M., Dimayuga, P.C., Zhou, J., Zhao, X., Yano, J., Trinidad, P., Honjo, T., et al. (2014) PLoS One. 9(3): p. e92095. 6. Jin, S., Zhou, F., Katirai, F. and Li, P.-L. (2011) Antioxidants & Redox Signaling. 15(4): p. 1043-1083. 7. Kabouridis, P.S., Janzen, J., Magee, A.L. and Ley, S.C. 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(2004) Applied Environmental Microbiology. 70(9): p. 5557-5568.   181  Appendices Appendix A   Bacterial strains, plasmids and oligonucleotides in Chapters 4 and 5 Strain or construct name Description  Reference Strains   RHA1 R. jostii RHA1 [254] ΔipdAB RHA1 RHA1 ΔRS22695-22690 This study ΔipdC RHA1 RHA1 ΔRS22685 This study ΔipdABC RHA1 RHA1 ΔRS22685-22690  This study ΔfadD3 RHA1 RHA1 ΔRS22410 [79] M. smegmatis mc2155  (NCBI 246196) ΔechA20 M. smegmatis mc2155 Δmsmeg_6001, HygR This study ΔechA20 M. smegmatis::echA20 mc2155 Δmsmeg_6001 containing pMVEchA20, HygR AprR This study ΔfadE32 M. smegmatis mc2155 Δmsmeg_6015, HygR This study ΔfadE32 M. smegmatis::fadE32 mc2155 Δmsmeg_6015 containing pMVFadE32, HygR AprR This study ΔipdF M. smegmatis mc2155 Δmsmeg_6011, HygR This study ΔipdF M. smegmatis::rv3559c mc2155 Δmsmeg_6011containing pMVrv3559c, HygR AprR This study ΔipdAB M.smegmatis mc2155 Δmsmeg_6002-6003, HygR This study Mtb M. tuberculosis Erdman (NCBI 652616) ΔipdAB Mtb Erdman Δerdman_3896-3897, HygR This study ΔipdAB Mtb::ipdAB Erdman Δerdman_3896-3897 containing pMVipdAB, HygR AprR This study ΔipdC Mtb Erdman Δerdman_3898, HygR This study ΔipdC Mtb::ipdABC Erdman Δerdman_3898 containing pMVipdABC, HygR AprR This study E. coli DH5α  Invitrogen - E. coli Rosetta2 pLysS  Novagen - 182  Strain or construct name Description  Reference E. coli BL21(DE3)  New England Biolabs - Plasmid constructs pK18mobsacB Plasmid for allelic exchange in RHA1. KanR [255] pK18∆ipdAB  pK18mobsacB with upstream region of RS22695 and downstream region of RS22690 cloned into EcoRI/HindIII sites. KanR This study pK18∆ipdC pK18mobsacB with upstream and downstream regions of RS22685 cloned into EcoRI/HindIII sites. KanR  pYUB854 Recombineering plasmid used for allelic exchange in mycobacteria. HygR [256] pYUBud6001 pYUB854 containing upstream (AflII/XbaI) and downstream (BglII/NheI) regions of Msmeg_6001 flanking hygR. HygR  This study pYUBud6011 pYUB854 containing upstream (AflII/XbaI) and downstream (BglII/NheI) regions of Msmeg_6011, flanking hygR. HygR This study pYUBud6015 pYUB854 containing upstream (AflII/XbaI) and downstream regions of Msmeg_6015, respectively, were introduced either side of hygR BglII/NheI (down). HygR This study pYUBudipdAB pYUB854 containing upstream (AflII/XbaI) region of Msmeg_6002 and downstream region of Msmeg_6003 (BglII/NheI) flanking hygR . HygR This study pYUBudipdC pYUB854 containing upstream  (AflII/XbaI) and downstream regions of Msmeg_6004, respectively, were introduced either side of hygR into pYUB854 using AflII/XbaI (up) and BglII/NheI (down). HygR This study pYUBudipdAB-mtb pYUB854 containing upstream (AflII/XbaI) region of rv3551 and downstream region of rv3552 (BglII/NheI) flanking hygR . HygR This study pYUBudipdC.mtb pYUB854 containing upstream (AflII/XbaI) region and downstream region of rv3553 (BglII/NheI) flanking hygR . HygR This study 183  Strain or construct name Description  Reference pMV361.apr Integrative plasmid with the constitutive hsp60 promoter for complementation in mycobacteria. AprR [257] pMVEchA20 pMV361.apr containing rv3550 inserted into the EcoRI/HindIII sites as a 766 bp fragment. A stop codon and RBS were added in the 5’UTR of the insert. AprR This study pMVFadE32 pMV361.apr containing msmeg_6015 inserted into the EcoRI/HindIII sites as a 988 bp fragment. A stop codon and RBS were added in the 5’UTR of the insert. AprR This study pMVrv3559c pMV361.apr containing rv3559c inserted into the EcoRI/HindIII sites as a 811 bp fragment. A stop codon and RBS were added in the 5’UTR of the insert. AprR This study pMVipdAB pMV361.apr containing rv3551-3552c inserted into the EcoRI/HindIII sites as a 1632 bp fragment. A stop codon and RBS were added in the 5’UTR of the transcript. AprR This study pMVechA20-ipdC pMV361.apr containing rv3550-3553c inserted into the EcoRI/HindIII sites as a 3536 bp fragment. A stop codon and RBS were added in the 5’UTR of the insert. AprR This study pET41b+ Expression vector for E. coli. KanR Novagen  pETRv3559 pET41b+ containing rv3559c (ipdF) with codons introducing an N-terminal His6 tag and TEVPro site following start codonas an 835 bp fragment inserted into NdeI/HindIII sites. KanR This study pTip-QC2 Expression vector for RHA1. Thiostrepton inducible promoter. CamR, AmpR [258] pTipR1EchA20 pTip-QC2 Expression vector for EchA20RHA1. 840 bp fragment containing RS27700 (echA20) with codons introducing a hexahistidine tag and TEVPro site following start codon. as an 840 bp fragment inserted into EcoRI/HindIII sites. CamR, AmpR This study 184  Strain or construct name Description  Reference pTipFadA6 pTip-QC2 containing rv3556c with codons introducing a hexahistidine tag and TEVPro site following start codon as a 1210 bp fragment inserted using EcoRI/HindIII into pTip-QC2. CamR, AmpR This study pTipR1IpdAB pTip-QC2 containing RS22695-22690 with codons introducing a hexahistidine tag and TEVPro site following start codon as a 1709 bp fragment inserted using EcoRI/HindIII into pTip-QC2. CamR,AmpR This study pTipIpdAB pTip-QC2 containing rv3551-2c with codons introducing a hexahistidine tag and TEVPro site following start codon inserted using EcoRI/HindIII into pTip-QC2. CamR,AmpR This study pTipRv3553 pTip-QC2containing rv3553 inserted using EcoRI/HindIII into pTip-QC2. CamR, AmpR This study pMAL-c2x Expression vector for E.coli used to make maltose binding protein (MBP)-fusion proteins. IPTG inducible promoter. AmpR (NEB #E8000S) pMALDOC21 Expression vector for MBP-IpdCDOC21 .1116 bp fragment containing DC0014_19. Introduced into pMAL-c2x using EcoRI/HindIII adding DC0014_19 in frame with MBP gene. AmpR This study          185   Appendix B  X-ray crystallography data collection and statistics B.1 KstR2: HIP-CoA crystallography statistics  PDB Code 4W97 Data collection  Space group C2 Cell dimensions    a, b, c, Å    β, °  72.5, 90.5, 49.8 129.7 Resolution, Å 25.00 – 1.60 Rmergea 0.032 (0.545)b I / (I) 48.68 (3.78) Completeness, % 100 (99.9) Redundancy 4.4 (4.2)   Refinement  Resolution, Å 24.85 – 1.60 No. of reflections: working, test 31023, 1650 R-factor/free R-factorc 16.0/19.9 (23.7/29.4) No. of refined atoms   Protein   Substrate   Solvent   Water  1626 64 4 310 B-factors, Å2  Protein   Substrate   Solvent   Water  28.9 37.9 41.2 43.5 r.m.s.d.   Bond lengths, Å   Bond angles,  0.02 2.14 aRmerge = ∑hkl ∑j | Ihkl,j - <Ihkl> | / ∑hkl ∑j Ihkl,j bValues in parentheses refer to highest resolution shells of 1.63-1.60 Å for data collection and 1.64-1.60 Å for refinement. cR-factor = ∑hkl | Fohkl – Fchkl | / ∑hkl Fohkl.  Free R-factor calculated with 5% reflections set aside.     186  B.2 IpdAB, IpdAB: COCHEA-CoA, and IpdAB E105AA: COCHEA-CoA crystallography statistics   IpdABRHA1 WT IpdABRHA1  E105AA·COCHEA-CoA IpdABRHA1  WT·COCHEA-CoA Wavelength 0.97948 0.97874 1.54 Resolution range 48.73  - 1.70 (1.76  - 1.70) 47.94  - 1.40 (1.45  - 1.40) 48.02  - 1.60 (1.66  - 1.60) Space group P 43212 P 43212 P 43212 Unit cell (Å, Å, Å, °, °, °) 68.91 68.91 241.37 90 90 90 69.17 69.17 241.87 90 90 90 69.29 69.29 241.86 90 90 90 Total reflections 522304 (51305) 819431 (75359) 837439 (27240) Unique reflections 65026 (6374) 116083 (11331) 77704 (6844) Multiplicity 8.0 (8.0) 7.1 (6.7) 10.8 (4.0) Completeness (%) 1.00 (1.00) 1.00 (0.99) 0.99 (0.89) Mean I/sigma(I) 16.54 (2.31) 20.62 (1.53) 31.31 (2.13) Wilson B-factor 18.06 16.09 21.51 R-merge 0.09076 (0.9778) 0.0541 (1.336) 0.0436 (0.4061) R-meas 0.09699 (1.044) 0.05838 (1.448) 0.04563 (0.4612) CC1/2 0.999 (0.818) 1.00 (0.624) 1.00 (0.934) CC* 1.00 (0.949) 1.00 (0.877) 1.00 (0.983) Reflections used in refinement 65021 (6374) 116047 (11327) 77599 (6823) Reflections used for R-free 3311 (347) 5804 (567) 3838 (357) R-work 0.1611 (0.2257) 0.1504 (0.2756) 0.1610 (0.4059) R-free 0.1916 (0.2508) 0.1716 (0.2766) 0.1886 (0.3955) CC(work) 0.971 (0.911) 0.978 (0.830) 0.973 (0.903) CC(free) 0.962 (0.893) 0.971 (0.776) 0.964 (0.880) Number of non-hydrogen atoms 4700 5014 4874 macromolecules 4148 4215 4183 ligands 79 78 78 Protein residues 551 543 542 RMS(bonds, Å) 0.006 0.006 0.006 RMS(angles, °) 0.83 0.92 0.93 Ramachandran favored (%) 97 97 98 Ramachandran allowed (%) 3 2.5 2.4 Ramachandran outliers (%) 0 0 0 Rotamer outliers (%) 0.23 0.45 0.23 Clashscore 1.56 2.01 3.23 Average B-factor (Å2) 23.6 25.4 27.0 macromolecules 21.3 22.0 24.3 ligands 71.7 66.3 71.9 solvent 35.7 40.4 39.6 Number of TLS groups 19 18 18         187  Appendix C  Analyses of metabolites CoASH    ɛ260nm, pH 7.0 = 11.9 mM-1 cm-1  [M+H]+ (ESI-MS/MS) 768.1210 (261.1269, 428.0364); C21H37N7O16P3S+ ; λmax= 258 nm 1H-NMR (850 MHz, D2O): δ= 8.69 (s,H,8), 8.29 (s,H,4), 6.19 (d,H,1’),4.85 (s,H,3’’), 4.59 (s,H,4’), 4.20 (s,2H,5’), 4.03 (s,2H,2’), 3.81-3.35 (m,8H), 2.59 (t,2H,6’’), 2.51 (t,2H,5’’), 0.90 (s,3H,10’’), 0.78 (s,3H,11’’)   13C-NMR (from 1H-13C HMBC/HSQC; 850 MHz, D2O):  δ= 178 (C4’’), 177 (C7’’), 158 (C6), 155 (C2), 153 (C4), 144 (C8), 122 (C5), 89 (C1’), 86 (C4’), 77, 75, 73, 68 (C5’c), 45 (C8’’), 41 (C2’’), 38 (C5’’c), 38 (C6’’c), 31 (C9’’c), 23 (C11’’), 21 (C10’’)   3aα-H-4α(carboxyl)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-OH HIC)        MP: 154.4-156oC (colorless liquid); λmax= 258 nm [M+H]+ (ESI-MS/MS) 962.2159 (455.2209, 428.0368); C32H51N7O19P3S+ 1H-NMR (400 MHz, MeOD): δ= 4.30-4.27 (m,H,5), 2.67 (dd, J =12.2,2.8 Hz,H,4), 2.48-2.41 (m,H,2), 2.31-2.20 (m,2H,3a,3), 2.16-2.09 (m,H,2), 1.84-1.80 (m,2H,6), 1.68-1.58 (m,2H,3,7), 1.53-1.49 (m,H,7), 0.91 (s,3H,8) 188  13C-NMR (100 MHz, MeOD): δ= 222 (C1), 177 (C9), 68 (C5), 49 (C7a), 48 (C4) 41 (C3a), 36 (C2), 30 (C6), 27 (C7), 24 (C3), 13 (C8) 2-TMS-5α-OH HIC GCMS Rt = 10.72 min. MS (70 eV, EI); m/z: 356 (4%), 341 (54%), 300 (11%), 147 (100%), 73 (78%) 3aα-H-4α(carboxyl)-5β-hydroxy-7aβ-methylhexahydro-1-indanone (5β-OH HIC)       MP: 193-195oC (colorless liquid); λmax= 258 nm [M+H]+ (ESI-MS/MS) 962.2170 (455.2209, 428.0367); C32H51N7O19P3S+  1H-NMR (400 MHz, MeOD): δ= 3.66 (ddd, J = 11.2, 10.0,5.5 Hz,H,5), 2.52-2.45 (m,2H,4,2), 2.21-2.11 (m,H,2), 1.96-1.89 (m,H,6), 1.86-1.67 (m,4H,3a,3,7), 1.65-1.57 (m,H,6), 1.41-1.35 (m,H,7), 0.97 (s,3H,8) 13C-NMR (100 MHz, MeOD): δ= 221 (C1), 177 (C9), 73 (C5), 52 (C4), 48 (C7a), 47 (C3a), 37 (C2), 31 (C6), 30 (C7), 23 (C3), 14 (C8) 2-TMS-5β-OH HIC GCMS Rt = 10.70 min. MS (70 eV, EI); m/z: 356 (9%), 341 (47%), 300 (15%), 147 (100%), 73 (96%) 2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA) Note: Carbon numbering used for NMR assignment differs from those used in Chapter 4 and 5  ɛ260nm, pH 7.0 = 16.4 mM-1 cm-1 (calculated).   [M+H]+ (ESI-MS/MS) 976.1960 (469.1994, 428.0358); C32H49N7O20P3S+; λmax= 250 nm 189  1H-NMR (850 MHz, D2O): δ= 8.64 (s,H,8c), 8.23 (s,H,4c), 6.19 (d,H,1’c),4.79 (s,H,3’’c), 4.63 (s,H,4’c), 4.23 (s,2H,5’c), 4.00 (s,2H,2’c), 3.58-3.39 (m,7H), 3.16 (m,2H, 9’’c), 2.73 (m,H, 3), 2.65 (m,2H, 5,1’), 2.59 (t,2H, 2’), 2.44 (t,H,5’’c), 2.38 (m,H,5), 2.10 (m,H,4), 1.81 (m,H,4) 1.23 (d 3J=9 Hz,3H,7), 0.90 (s,3H,10’’c), 0.78 (s,3H,11’’c);  c denotes CoA moiety  13C-NMR (from 1H-13C HMBC/HSQC; 850 MHz, D2O):  δ= 203 (C6), 201 (C1’’), 182 (C3’), 177 (C4’’c), 177 (C7’’c), 175 (C2), 156 (C6c), 155 (C2c), 152 (C4c), 142 (C8c), 139 (C1), 121 (C5c), 89 (C1’c), 83 (C4’c), 77 (C3’’c), 75 (C2’c), 73 (C3’c), 68, 62 (C5’c), 41 (C2’’c), 38 (C5’’c), 38 (C6’’c), 36 (C5), 36 (C1’), 35 (C3), 32 (C2’), 31 (C9’’c), 31 (C4), 24 (C11’’c), 21 (C10’’c), 19 (C7);  c denotes CoA moiety  4-Methyl-5-oxo-octanedioc acid (MOODA) 1H NMR (850 MHz, D2O): δ= 2.97 (m,1H,6x), 2.89 (m,1H,6y), 2.77 (m,1H,4), 2.61 (t,2H,8), 2.37 (t,2H,2), 1.94 (m,1H,3x), 1.69 (m,1H,3y), 1.13 (d,3J= 7.1 Hz,3H, 9)  13C NMR (from 1H-13C HMBC/ HSQC; 850 MHz, D2O): δ= 221 (C5), 180 (C1), 179 (C8), 48 (C4), 38 (C6), 32 (C2), 30 (C7), 29 (C3), 18 (C9)  2-TMS-MOODA GCMS Rt = 10.02 min. MS (70 eV, EI); m/z: 346 (1%), 331 (12%), 241 (14%), 173 (100%), 125 (32%), 73 (82%)  4-Methyl-5-oxo-octanedioyl-CoA (MOODA-CoA) CoASOOO-O  [M+H] 952.1960 (445.1999, 428.0371) m/z; C30H49N7O20P3S+; λmax = 258 nm  1H-NMR (600 MHz, D2O): δ(ppm) = 8.53 (s, 1H), 8.24 (s, 1H), 6.2 (d, 1H), 4.65 (s, 1H), 4.2 (s, 2H), 4.0 (s, 2H), 3.8 (m, 1H), 3.6-3.5 (m, 2H), 3.50 (q, 2H), 3.49 (m, 3H), 3.30-3.45 (m, 4H), 2.95 (q, 2H), 2.83 (m, 1H), 2.73 (m, 1H), 2.68 (m, 1H), 2.58 (q, 2H), 2.3-2.4 (m, 4H), 1.64 (m, 1H), 1.05 (d, 3H), 0.86 (s, 3H), 0.72 (s, 3H)      190  2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1 1-TMS-COCHE GCMS Rt = 9.32 min. MS (70 eV, EI); m/z: 254 (83%), 239 (25%), 221 (59%), 197 (40%), 137 (92%), 122 (100%) 1H-NMR (600 MHz, D2O): δ= 5.8 (s,1H,1), 2.6 (m,6H,5,3’,1’), 2.4 (m,1H,3), 2.1 (m,1H,4x), 1.8 (m,1H,4y), 1.2 (d,3J= 8 Hz,3H,7) 13C-NMR (from HSQC/HMBC; 600 MHz, D2O): δ= 205 (C6), 180 (C3’), 174 (C2), 128 (C1), 36 (C5), 34 (C3), 34 (C2’), 32 (C4), 32 (C1’),  17 (C7)  (7aS)-7a-Methyl-1,5-dioxo-2,3,5,6,7,7a-hexahydro-1H-indene-4-carboxyl-CoA (HIEC-CoA)   [M+H] 958.1899 (451.1914, 428.0378) m/z; C32H47N7O19P3S+; λmax = 253 nm    

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