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View of the Tetrapyrrole Biosynthesis Pathway with Its Substrates, Major Intermediates and Products

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View of the Tetrapyrrole Biosynthesis Pathway with Its Substrates, Major Intermediates and Products Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2019 Siroheme Biosynthesis and Regulation of SJosierpho Mhasetemrs Peen nDingetoni IcV iency Follow this and additional works at the DigiNole: FSU's Digital Repository. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES SIROHEME BIOSYNTHESIS AND REGULATION OF SIROHEME DEFICIENCY By JOSEPH MASTERS PENNINGTON IV A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2019 Joseph Masters Pennington IV defended this dissertation on April 12, 2019. The members of the supervisory committee were: Hong Li Professor Directing Dissertation Kathryn M. Jones University Representative M. Elizabeth Stroupe Committee Member Michael Blaber Committee Member Wei Yang Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements. ii This dissertation is dedicated to my parents, Joe and Patricia; Your love and support have made this possible. iii ACKNOWLEDGMENTS I would like to initially thank my PI, Beth Stroupe, for all her support, patience, and training over the past years. The collaborative atmosphere of the lab and encouragment of undergradaute and high school students to pursue research played a large role in my develpoment as an independent researcher and a future mentor. I would also like to thank my committee, Hong Li, Kathryn Jones, Michael Blaber, and Wei Yang for their input and feedback during each step of my PhD journey. A huge thank you to the FSU core facilites, specifically Cladius Mundoma, Soma Sundaram, Joan Hare, Brian Washburn, Cheryl Pye, and Kristina Poduch. Much of this work would have been impossible, or at best much harder then needed, without your work, guidance, and advice. Next, I want to thank my current and former lab mates, Daniel Murray, Mike Kopylov, Isabel Askenasy, Matt Johnson, Jay Rai, Rachel Andrews, Marisa Cepeda, Lauren McGarry, Angela Tavolieri, Trent Quist, and Ashley Santiago. It has been an honor working and learning alongside of each of you. Lastly, I want to thank my family. All of your love and support as I pursued this crazy degree has meant more than I can ever show. iv TABLE OF CONTENTS LIST OF TABLES .................................................................................................................. viii LIST OF FIGURES .................................................................................................................. ix ABSTRACT ...............................................................................................................................x CHAPTER 1 INTRODUCTION .................................................................................................1 1.1. Preface ..........................................................................................................................1 1.2. Anatomy of a tetrapyrrole .............................................................................................1 1.3. Tetrapyrrole biosynthesis ..............................................................................................2 1.4. The discovery of siroheme and the siroheme biosynthetic pathway ...............................4 1.5. Siroheme biosynthesis in different organisms ................................................................5 1.6. Siroheme in sulfite and nitrite reductases ......................................................................5 CHAPTER 2 THE DEHYDROGENASE AND CHELATASE MECHANISMS OF SALMONELLA TYPHIMURIUM SIROHEME SYNTHASE .......................................................7 2.1. Introduction ..................................................................................................................7 2.2. Materials and methods ..................................................................................................8 2.2.1. Materials and chemicals .........................................................................................8 2.2.2. Cloning of StCysG mutants ....................................................................................8 2.2.3. Protein expression and purification ........................................................................8 2.2.4. Structure determination ..........................................................................................9 2.2.5. Substrate biosynthesis ............................................................................................9 2.2.6. Anaerobic crystal soaking and freezing ................................................................ 10 2.2.7. Data collection and processing ............................................................................. 10 2.2.8. In vivo complementation assays of StCysG mutants ............................................. 10 2.2.9. In vitro specific activity enzyme assays ................................................................ 10 2.3. Results ........................................................................................................................ 11 2.3.1. Structure determination of CysG bound to PC2 + NADH, SHC, and Co-SHC and CysG variants .................................................................................................................... 11 2.3.2. In vivo activity assays of StCysG mutants ............................................................ 11 2.3.3. In vitro activity assays of StCysG mutants ........................................................... 11 2.4. Discussion................................................................................................................... 12 2.4.1. PC2 is puckered in the CysGB active site .............................................................. 12 2.4.2. Coordination of SHC in CysGB ............................................................................ 12 2.4.3. Co-SHC is weakly bound in the second CysGB active site .................................... 13 2.4.4. Fluorescence of CysG substrates in vivo .............................................................. 13 2.4.5. Conservation with BmSirC .................................................................................. 13 v CHAPTER 3 IDENTIFICATION AND CHARACTERIZATION OF MTCYSG AND CHE1, TWO ENZYMES RESPONSIBLE FOR SIROHEME PRODUCTION IN MYCOBACTERIUM TUBERCULOSIS ...................................................................................................................... 25 Introduction ................................................................................................................ 25 Materials and methods ................................................................................................ 26 3.2.1. Materials and chemicals ....................................................................................... 26 3.2.2. Identification of tuberculosis CysG candidates ..................................................... 27 3.2.3. Cloning of CysG Candidates ................................................................................ 27 3.2.4. In vivo CysG complementation assays ................................................................. 27 3.2.5. Protein expression and purification of MtCysG and Che1 .................................... 27 3.2.6. Substrate biosynthesis .......................................................................................... 28 3.2.7. In vitro activity assays of MtCysG and Che1 ........................................................ 28 3.2.8. Crystallization of MtCysG and diffraction ............................................................ 29 Results and discussion................................................................................................. 29 3.3.1. MtCysG is located in an operon associated with vitamin B12 synthesis................ 29 3.3.2. MtCysG is missing the alpha-helical bundle domain ............................................ 30 3.3.3. MtcysG does not complement cysG-deficient E. coli ........................................... 30 3.3.4. MtCysG is an NAD+-dependent PC2 dehydrogenase but not a ferrochelatase....... 30 3.3.5. Diffraction of MtCysG crystals ............................................................................ 31 CHAPTER 4 THE N-TERMINAL DOMAIN OF ESCHERICHIA COLI ASSIMILATORY NADPH-SULFITE REDUCTASE HEMOPROTEIN IS AN OLIGOMERIZATION DOMAIN THAT MEDIATES HOLOENZYME ASSEMBLY .................................................................. 37 4.1. Introduction .................................................................................................................... 37 4.2. Materials and methods .................................................................................................... 39 4.2.1. Recombinant protein production: SiRHP ................................................................. 39 4.2.2. Recombinant protein production: SiRFP .................................................................. 39 4.2.3. Recombinant protein production: SiRFPFMN............................................................. 40 4.2.4. Recombinant protein production: SiRFP43................................................................ 40 4.2.5. Recombinant protein production: SiR ...................................................................... 41 4.2.6. SiR, SiRFP43,
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