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Exploring the Functional Roles of Π-Helices and Flavin-Induced Oligomeric State Changes in Flavin Reductases of Two-Component Monooxygenase Systems

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Exploring the Functional Roles of Π-Helices and Flavin-Induced Oligomeric State Changes in Flavin Reductases of Two-Component Monooxygenase Systems Exploring the Functional Roles of π-helices and Flavin-Induced Oligomeric State Changes in Flavin Reductases of Two-Component Monooxygenase Systems by Jonathan Makau Musila A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 6, 2017 Keywords: Oligomerization, kinetics, desulfonation, π-helix, dis(asso)ciation Copyright 2017 by Jonathan Makau Musila Approved by Holly R. Ellis, Chair, Professor of Chemistry and Biochemistry Douglas C. Goodwin, Associate Professor of Chemistry and Biochemistry Evert C. Duin, Associate Professor of Chemistry and Biochemistry Steven Mansoorabadi, Assistant Professor of Chemistry and Biochemistry Abstract Sulfur-containing biomolecules participate in various chemical and structural functions in enzymes. When sulfate is limiting, bacteria upregulate ssi enzymes to utilize organosulfonates as an alternative source. The alkanesulfonate monooxygenase enzymes mitigate sulfur scarcity through desulfonation of various alkanesulfonates releasing sulfite which is incorporated into sulfur-containing biomolecules. This two-component monooxygenase system utilizes flavin as a substrate with the SsuE enzyme supplying reduced flavin to SsuD. It is unclear what structural properties of flavin reductases of two-component systems dictate catalysis. The SsuE enzyme undergoes a tetramer to dimer oligomeric switch in the presence of FMN. Oligomeric state changes are common in flavin reductases but the roles and regulation of the quaternary structural changes have not been evaluated. Intriguingly, the flavin reductases of two-component systems contain π- helices located at the tetramer interface. π-Helices are generated by a single amino acid insertion in an established α-helix to confer an evolutionary advantage. Substitution of the π-helix insertional residue in SsuE (Tyr118) generated FMN-bound Y118A and Y118S SsuE variants, unlike the wild-type enzyme which is flavin-free. Structural and kinetic analyses were performed on these canonical flavoprotein variants and on the flavin-free variants (Y118F and ΔY118 SsuE) to understand the roles of π-helix in SsuE. We further investigated the structural effects of perturbing the π-helix in SsuE by solving the three- dimensional structures of Y118A SsuE in the oxidized form, when reduced, and in the apo form. Our findings show the π-helix is vital in the functioning of SsuE as a flavin reductases of two- ii component systems. In combination, the studies suggested the π-helix in SsuE promotes structural stability through hydrogen bonding and π-stacking interactions, and facilitates the transfer of reduced flavin to SsuD through protein-protein interactions. The π-helix may regulate the oligomeric changes in SsuE upon flavin binding and could be vital in controlling dioxygen reactivity. We also report herein that reduced flavin transfer from SsuE to SsuD occurs through a channeling mechanism facilitated by conformational changes in the flexible loop of SsuD. Overall, these studies unravel the evolutionary role of the π-helices in defining oligomeric state changes and in the transfer of reduced flavin in two-component monooxygenase systems. iii Acknowledgments A time has come for me to wind up my graduate studies and unfold a new chapter of my life. This journey would not have been a success without a team’s effort. I am sincerely indebted to Prof. Holly R. Ellis for the opportunity to do research in her lab, coupled with her mentorship throughout my graduate studies here in Auburn University. I am particularly grateful for her scientific and research input, and for providing the resources which made this research a success. I also appreciate collective and individual contributions of Dr. Douglas C. Goodwin, Dr. Evert C. Duin, and Dr. Steven Mansoorabadi for ensuring that I remained on track during my graduate studies and research, for advising constructively, and for allowing me access to the instrumentation in their respective labs. In addition, I highly appreciate the role played by Dr. Floyd Woods as the University Reader. I also recognize former and current lab mates among them Dr. Catherine Njeri, Paritosh Dayal, Dianna Forbes, Claire Graham, Katie Stanford, and Richard Hagen for open discussions and scientific opinions during my stay in CB351. I also express my gratitude to the Department of Chemistry and Biochemistry Auburn University for offering me with the opportunity to advance my academic career, and the National Science Foundation for financing this research project. Finally, words cannot express enough the appreciation I have for the sacrifice, the background perseverance, physical, and emotional support invested by my lovely wife Naomi Makau, and our adventurous boys Wesley Makau and Johnstone Makau. Indeed, your investment was not in vain! This dissertation work is dedicated to my dad, John Musila who has sacrificed his entire life to see me this far! Long live Auburn University! War Eagle! iv Table of Content Abstract ......................................................................................................................................... ii Acknowledgments........................................................................................................................ iv List of Tables ................................................................................................................................ x List of Schemes ............................................................................................................................ xi List of Figures ............................................................................................................................. xii CHAPTER ONE ............................................................................................................................. 1 LITERATURE REVIEW ............................................................................................................... 1 1.1 Sulfur Metabolism in Bacteria ......................................................................................... 1 1.1.1 General Introduction to Sulfur Metabolism .............................................................. 1 1.1.2 Sulfur Assimilation Pathway in Bacteria .................................................................. 3 1.1.3 Persulfides in Trafficking of Sulfur in Biosynthetic Pathways ................................ 5 1.1.4 Sulfur Incorporation and Occurrence in Biomolecules .......................................... 10 1.1.5 Scavenging Sulfur during Sulfur Starvation Conditions ........................................ 13 1.1.6 The Alkanesulfonates Transport System ................................................................ 17 1.1.7 Regulation of the ssi Operons ................................................................................. 18 1.2 Flavoproteins in Enzymology ........................................................................................ 21 1.2.1 Flavin ...................................................................................................................... 21 1.2.2 Biosynthesis of Flavins in Bacteria......................................................................... 23 v 1.2.3 Spectral Properties of Flavin at Different Oxidation States .................................... 26 1.2.4 Flavoproteins........................................................................................................... 29 1.2.5 Reactivity of Flavin with Molecular Oxygen ......................................................... 30 1.3 Two-Component Flavin-Dependent Systems ................................................................ 35 1.3.1 Examples of Two-Component Flavin-Dependent Systems .................................... 35 1.3.2 Kinetic Analysis of the Flavin Reductases and Monooxygenase of Two- Component Monooxygenase Systems ................................................................................... 40 1.3.3 Structural Properties of the Two-Component Flavin-Dependent Enzymes ........... 48 1.4 π-helices in Proteins ....................................................................................................... 62 1.4.1 Introduction to Helical Structures in Proteins ......................................................... 62 1.4.2 Generation of π-Helices in Protein ......................................................................... 65 1.4.3 π-helices Augment Function in Proteins ................................................................. 67 1.4.4 π-helices show Preference for Certain Amino Acids .............................................. 70 1.4.5 Assignment of π-helices in Secondary Structures of Proteins ................................ 72 1.5 Protein Oligomerization ................................................................................................. 73 1.5.1 Importance of Protein Oligomerization .................................................................. 73 1.5.2 Oligomeric State Changes in Flavin Reductases .................................................... 75 1.6 Summary ........................................................................................................................ 77 CHAPTER TWO .......................................................................................................................... 81 2 Transformation of a Flavin-Free FMN Reductase to a Canonical Flavoprotein through Modification
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