STRUCTURAL AND MECHANISTIC CHARACTERIZATION OF ENZYMES IN PERSULFIDE OXIDATION AND MONOLIGNOL BIOSYNTHESIS PATHWAYS By STEVEN ANDREW SATTLER A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY School of Molecular Biosciences MAY 2018 © Copyright by STEVEN ANDREW SATTLER, 2018 All Rights Reserved © Copyright by STEVEN ANDREW SATTLER, 2018 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of STEVEN ANDREW SATTLER find it satisfactory and recommend that it be accepted. ChulHee Kang, Ph.D., Chair Susan Wang, Ph.D. Luying Xun, Ph.D. Margaret Black, Ph.D. ii ACKNOWLEDGMENT There are many people who I would like to thank for helping place me in a position to earn this degree. Foremost, I would like to thank my academic advisors, friends, and family for their guidance, support, and patience while I spent several years as an undergraduate and doctoral student. Most of my achievements are attributed to your respective influences on my education or disposition, and for that I will always be grateful. Even if you are not mentioned by name in this acknowledgment, know that your influence is appreciated. I would first like to thank my doctoral advisor, Dr. ChulHee Kang, for providing me with an opportunity to earn this degree. His guidance and willingness to listen to my ideas were crucial to my development as a scientist and prepared me for a professional career in theoretical work. I would also like to thank Dr. Luis Matos, who provided me with my first research opportunity as an undergraduate. I partially owe any success that I have had as a graduate student to his guidance. The final academician I would like to recognize by name is Susan Wick Johnson. As a teacher and friend, she was the first person to guide me in understanding the rewards of scholastic success. To members of my graduate research group, both past and present, I would like to thank Abigail Green, Robert Hayes, Kevin Lewis, Timothy Moural, Alexander Walker, Se-Young Jun, and Shin-Hwa Wu. The alliances we have developed through the years are both personal and professional in nature, and I would like to thank all of you for your friendship and collaboration as we were all trying to make difficult transitions in our lives. I will never forget the impact that that our relationships had and continue to have on my development as a scientist and person. To my family and close friends, the ways and magnitudes of how you have affected my life for the better are immeasurable. I would like to thank my parents, Scott and Stacie Sattler and Roni and Bill Jarrell, my grandparents Richard and Sandy Stone and Edith and Larry Sattler, and iii my siblings Chris Sattler, Brian Sattler, Brook Sattler, J.R. Jarrell, Carlee Jarrell, Austin Sattler, and Corey Jarrell. I would also like to thank April Frers, Rahul Dhal, Rob Jones, Josh Swider, Justin Johnson, Jason Price, Deborah Bohnen, Enrique Alvarado, and Brett Vanderwurff. I have always greatly appreciated your friendship, love and support, and have learned valuable lessons from each of you. Finally, I would like to thank the administrative staff of the School of Molecular Biosciences, particularly Kelly McGovern and Tami Breske. I never missed a deadline because of them. iv STRUCTURAL AND MECHANISTIC CHARACTERIZATION OF ENZYMES IN PERSULFIDE OXIDATION AND MONOLIGNOL BIOSYNTHESIS PATHWAYS Abstract by Steven Andrew Sattler, Ph.D. Washington State University May 2018 Chair: ChulHee Kang We conducted investigations of the structural and biochemical properties of two distinct types of enzyme from different pathways: cinnamoyl-CoA reductases (CCRs) and the persulfide dioxygenases (PDOs). CCR is an enzyme of the monolignol biosynthesis pathway in plants that uses NADPH to reduce any of three major hydroxycinnamoyl-CoA thioesters, thus catalyzing the formation of hydroxycinnamaldehydes. These aldehydes are further reduced by cinnamyl alcohol dehydrogenase (CAD) to form the alcohol substrates required for lignin biosynthesis. One CCR, SbCCR1, was crystallized in the presence of NADPH and the structure was determined at 2.9 Å resolution. It was determined—through site-directed mutagenesis, ITC, molecular docking, and kinetics assays—that not only is feruloyl-CoA the preferred substrate for SbCCR1, but residues Thr154 and Tyr310 are essential to binding functional groups about the aromatic ring of the substrate. Production of a T154Y mutant of SbCCR1 resulted in significant reduction in substrate preference for feruloyl-CoA over p-coumaroyl-CoA, providing support for the hypothesis that substitutions can be made at this position leading to favorable reductions in or softening of lignin v content in S. bicolor. Furthermore, confirmed the existence of an additional CCR in S. bicolor through multiple-sequence alignment and kinetics analyses. PDOs catalyze the oxidation of a sulfane sulfur on glutathione persulfide (GSSH) or higher glutathione polysulfanes (GSSnH, where n > 1) during the process of detoxifying dihydrogen sulfide (H2S). In this study, the structures of PDOs from M. xanthus (MxPDO1) and P. putida (PpPDO2) were determined in the presence of a catalytic iron, as well as in the presence of both iron and product molecule glutathione for the PDO from P. putida. Structure alignments with other PDOs, in addition to the 1.46 Å PpPDO2/GSH complex structure, revealed key substrate-binding residues and provided a basis for activity differences between PDOs and a closely related group of enzymes, the glyoxalases II. Static light scattering further showed that, despite high identity to the glyoxalases II, PDOs are likely dimeric. In summary, we described interactions between PDOs and GSH, as well as provided information as to how two highly related groups of enzymes could differ in form and function. vi TABLE OF CONTENTS Page ACKNOWLEDGMENT................................................................................................................ iii ABSTRACT .....................................................................................................................................v LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES .........................................................................................................................x CHAPTERS CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 General Overview ....................................................................................................1 1.2 Function of CCR ......................................................................................................1 1.3 Mutations to Monolignol Biosynthetic Enzymes and the “Lignin Problem” ..........1 1.4 Function of PDOs ....................................................................................................2 1.5 PDOs and Ethylmalonic Encephalopathy (EE) .......................................................3 1.6 Structural and Evolutionary Relationships between PDOs and the Glyoxalases II ................................................................................................................3 1.7 Chapter Summaries ..................................................................................................5 1.8 References ..............................................................................................................10 CHAPTER TWO: CHARACTERIZATIONS OF TWO PERSULFIDE DIOXYGENASES OF THEMETALLO-β-LACTAMASE SUPERFAMILY .................14 2.1 Contributions..........................................................................................................14 2.2 Abstract ..................................................................................................................14 2.3 Introduction ............................................................................................................15 2.4 Materials and Methods ...........................................................................................17 2.5 Results ....................................................................................................................21 vii 2.6 Discussion ..............................................................................................................24 2.7 Acknowledgments..................................................................................................28 2.8 References ..............................................................................................................43 CHAPTER THREE: STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF CINNAMOYL-COA REDUCTASES ........................................................................47 3.1 Contributions..........................................................................................................47 3.2 Abstract ..................................................................................................................47 3.3 Introduction ............................................................................................................48 3.4 Materials and Methods ...........................................................................................51 3.5 Results ....................................................................................................................56 3.6 Discussion