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BIOSYNTHETIC and FUNCTIONAL STUDIES of BACILLITHIOL in GRAM- POSITIVE BACTERIA by ZHONG FANG a Dissertation Submitted to The

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BIOSYNTHETIC AND FUNCTIONAL STUDIES OF BACILLITHIOL IN GRAM- POSITIVE BACTERIA BY ZHONG FANG A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Chemistry May 2015 Winston-Salem, North Carolina Approved By: Patricia C. Dos Santos, Ph.D., Advisor H. Alexander Claiborne Jr, Chair Rebecca W. Alexander, Ph.D. Stephen Bruce King, Ph.D. Mark E. Welker, Ph.D. TABLE OF CONTENTS LIST OF ILLUSTRATIONS AND TABLES ....................................................... iv LIST OF ABBREVIATIONS ............................................................................... viii ABSTRACT .......................................................................................................... ix CHAPTER 1 Introduction ..................................................................................... 1 1.1 Biothiol ........................................................................................................ 1 1.2 Cysteine ...................................................................................................... 2 1.3 Glutathione .................................................................................................. 6 1.4 Mycothiol ................................................................................................... 10 1.5 Bacillithiol .................................................................................................. 17 CHAPTER 2 Cross-functionalities of Bacillus deacetylases involved in bacillithiol biosynthesis and bacillithiol-S-conjugate detoxification pathways ...................... 25 Abstract ........................................................................................................... 26 Introduction ..................................................................................................... 27 Method and materials ...................................................................................... 30 Results ............................................................................................................ 37 Discussion ....................................................................................................... 51 CHAPTER 3 Protective role of bacillithiol in superoxide stress and metal homeostasis in Bacillus subtilis .......................................................................... 56 Abstract ........................................................................................................... 57 Introduction ..................................................................................................... 58 Method and materials ...................................................................................... 62 Results ............................................................................................................ 69 Discussion ....................................................................................................... 86 ii CHAPTER 4 Characterization of NifS cysteine desulfurase and its role on NAD biosynthesis in Bacillus subtilis ........................................................................... 90 Introduction ..................................................................................................... 91 Method and materials ...................................................................................... 99 Results .......................................................................................................... 104 Discussion ..................................................................................................... 115 CHAPTER 5 Functional Studies of Nfu in Bacillus subtilis ............................... 119 Introduction ................................................................................................... 120 Method and materials .................................................................................... 124 Results .......................................................................................................... 128 Discussion ..................................................................................................... 132 CHAPTER 6 CONCLUDING REMARKS ......................................................... 135 REFERENCES ................................................................................................. 139 SCHOLASTIC VITA ......................................................................................... 155 iii LIST OF ILUSTRATIONS AND TABLES FIGURES CHAPTER 1 Figure 1.1 Common low-molecule-weight thiols found in living organisms. 1 Figure 1.2 Mechanism of oxidoreductase-catalyzed formation of disulfide bonds. 3 Figure 1.3 The common cysteine oxidation states in biological systems. 4 Figure 1.4 Common metal-thiol cofactors found in biological enzymes. 6 Figure 1.5 The catalytic cycle of glutathione reductase. 9 Figure 1.6 Biosynthetic pathway of mycothiol. 12 Figure 1.7 Detoxification pathways of fosfomycin through FosA, FosB and FosX. 21 CHAPTER 2 Figure 2.1 The proposed biosynthetic pathway and detoxification mechanism of 27 bacillithiol. Figure 2.2 Substrate saturation curves of BA1557, BC1534, BA3888 and BC3461. 39 Figure 2.3 Sequence identity (grey boxes) and similarity (white boxes) between the N- deacetylase enzymes 40 Figure 2.4 Drawgram of deacetylase enzymes 40 Figure 2.5 Far-UV circular dichroism spectra of deacetylases 41 Figure 2.6 Active site region of B. cereus BC1534 (PDB Accession 2IXD). The Zn2+ is 41 coordinated with His12, Asp15 and His113. Figure 2.7 Amino acid sequence alignment of deacetylase enzymes. 42 Figure 2.8 Deacetylation activity of apo-BA1557 reconstituted with different 43 stoichiometric ratio of Zn2+. Figure 2.9 Activity pH profiles deacetylase enzymes. 45 Figure 2.10 Activity pH profiles deacetylase activity of BA1557. 46 iv Figure 2.11 Proposed mechanism for BshB deacetylation reaction 46 Figure 2.12 Model of the active site of BC1534 (PDB code 2IXD) in complex with 49 GlcNAc-Mal. CHAPTER 3 Figure 3.1 Growth profiles of B. subtilis wild type, ∆bshA, and ∆bshC AmyE:Pxyl-bshC strains in LB and Spizizen’s MM plus leu, Isoleu, Gln and Glu. 70 Figure 3.2 Growth profile of B. subtilis strain lacking BSH in MM. 71 Figure 3.3 Activities of the Fe-S proteins glutamate synthase (GOGAT), aconitase (ACN), isopropyl malate isomerase (LeuCD) and the non-Fe-S protein malate dehydrogenase (MDH) in cell lysates of B. subtilis wild type and ΔbshA strain. 73 Figure 3.4 Activities of mononuclear iron enzyme threonine dehydrogenase and quercetin 2,3-dioxygenase. 75 Figure 3.5 Growth curves of wild-type B. subtilis and ∆bshA strain in MM with 0.05% casamino acid in presence of various stress challenges. 77 Figure 3.6 Cellular BSH redox status upon metal and superoxide stress. 78 Figure 3.7 Specific activities of ACN (A) and GOGAT (B) under stress conditions. 80 Figure 3.8 Total iron and manganese concentration analysis in wild-type B. subtilis and ∆bshA strain after exposure to 100 µM of paraquat stress. 82 Figure 3.9 MnSOD activity in the cell lysates of CU1065 (wt) and HB11002 (bshA) before and 30 min after the treatment of 100 µM paraquat. 83 Figure 3.10 Western blot analysis of Fe-S biosynthetic enzymes 83 CHAPTER 4 Figure 4.1 De novo and salvage NAD biosynthetic pathways. 92 Figure 4.2 Biosynthetic conversion steps in the formation of quinolinic acid. 94 Figure 4.3 The nicotinic acid de novo gene region. 95 Figure 4.4 In-vitro NadA activation. 96 v Figure 4.5 NifS cysteine desulfurase reaction. 97 Figure 4.6 Cysteine saturation curves of NifS in terms of alanine and sulfide produced. 105 Figure 4.7 A. pH-activity profile of cysteine desulfurase NifS. 106 Figure 4.8 Cysteine desulfurase activities of NifS under various conditions. 108 Figure 4.9 Far-UV circular dichroism spectra of NadA+NadB, NifS+NadA, NifS+NadB, and NifS+NadA+NadB. 109 Figure 4.10 Analytical gel filtration chromatograms of NifS, NadA, NadB and different combinations of the three enzymes. 111 Figure 4.11 UV-visible absorption spectra of as isolated B. subtilis NadA, after anaerobic treatment with cyanide and EDTA and after reconstitution. 112 Figure 4.12 Reconstitution of apo-NadA in presence of various sulfur sources under reducing and non-reducing condition. 113 Figure 4.13 Reconstitution of apo-NadA in presence of equivalent NadB under reducing (A) and non-reducing (B) conditions. 114 Figure 4.14 Reconstitution of apo-NadA with Fe and sulfide in presence of 2 mM DTT, BSH or none. 115 CHAPTER 5 Figure 5.1 Biosynthetic scheme of Fe-S cluster. 120 Figure 5.2 Schematic representations of domain structures of NifU, NfuA, IscA, Nfu, and IscU proteins from bacteria. Azotabacter vinelandii (Av), E. coli (Ec), B. subtilis (Bs) and S. aureus (Sa). 122 Figure 5.3 Sequence alignments of B. subtilis Nfu with E.coli NfuA U domain and A.vinelandii NfuA U domain. 123 Figure 5.4 A. Complementation analysis of B.subtilis Nfu in wild type (MG) and ΔnfuA E. coli strains under oxidative stress and iron starvation conditions. 129 vi Figure 5.5 Enzymatic activities of GOGAT, ACN, and MDH in MG1655 and ∆NfuA strains containing different plasmids. 130 Figure 5.6 UV-visible absorption spectra of as isolated Nfu, apo-Nfu and reconstituted. 131 Figure 5.7 A. apo-AcnA was incubated with reconstituted Nfu at room temperature under anaerobic conditions, and aliquots from different time points were taken for activity assays as described in Method and Materials.
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