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Mechanistic Studies on D-Arginine Dehydrogenase and Functional Annotation of a Novel NADH:Quinone Oxidoreductase (PA1024)

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Mechanistic Studies on D-Arginine Dehydrogenase and Functional Annotation of a Novel NADH:Quinone Oxidoreductase (PA1024) Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry 8-7-2018 Mechanistic Studies on D-arginine Dehydrogenase and Functional Annotation of a Novel NADH:quinone Oxidoreductase (PA1024) Jacob A. Ball Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Recommended Citation Ball, Jacob A., "Mechanistic Studies on D-arginine Dehydrogenase and Functional Annotation of a Novel NADH:quinone Oxidoreductase (PA1024)." Dissertation, Georgia State University, 2018. https://scholarworks.gsu.edu/chemistry_diss/148 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. MECHANISTIC STUDIES ON D-ARGININE DEHYDROGENASE AND FUNCTIONAL ANNOTATION OF A NOVEL NADH:QUINONE OXIDOREDUCTASE (PA1024) by JACOB BALL Under the Direction of Giovanni Gadda, PhD ABSTRACT Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is an FAD-dependent enzyme that catalyzes the oxidative deamination of D-arginine to generate the corresponding α- keto acid and ammonia. The enzyme is similar to D-amino acid oxidase, except that PaDADH is a strict dehydrogenase with no oxygen reactivity. The enzyme exhibits broad substrate specificity and can oxidize 17 of the 20 common amino acids. The best substrates for the enzyme are D-arginine and D-lysine based on the second order rate constant kcat/Km values. The 3D structure of the DADH-iminoarginine complex suggests that E87 engages in an electrostatic interaction with the positively charged guanidinium group of D-arginine. The 3D structure also displays a hydrogen bonding network of water molecules that connects H48 to the substrate α-amine, suggesting it may serve as catalytic base. E87 and H48 were mutated to produce E87L and H48F variants, and pH effect kinetic approaches with zwitterionic and cationic substrates were employed. The data, in combination with previous results on Y53 and Y249 variants, suggests that there is no catalytic base, since the catalytic pKa is present in all variants. The results are also consistent with E87 required to be deprotonated to bind cationic substrates. In the second part of this dissertation, a presumed nitronate monooxygenase from P. aeruginosa, PA1024, is characterized. PA1024 is determined to not possess NMO activity and instead catalyze the two-electron reduction of quinones via the oxidation of NADH. The enzyme has a strict preference for NADH over NADPH. The functional annotation and bioinformatics identifies a novel class of FMN-dependent NADH:quinone oxidoreductases (NQO) with a TIM- barrel fold. The pH effects reveal that PA1024 has two regimes of activity at low and high pH dictated by the deprotonation of an enzymatic residue with pKa ~7. In addition, the 3D X-ray structure of PA1024 in complex with the reaction product NAD+ is solved. The structure reveals the structural basis for the strict NADH specificity, which relies on a steric constraint imposed by a nearby P78 and Q80. The structure also reveals a conformational gating mechanism of Q80 and an unusual interrupted helix structural pattern to bind the pyrophosphate of NAD+. INDEX WORDS: flavin, oxidoreductase, NADH, quinones, D-arginine dehydrogenase, pH effects, enzyme kinetics MECHANISTIC STUDIES ON D-ARGININE DEHYDROGENASE AND FUNCTIONAL ANNOTATION OF A NOVEL NADH:QUINONE OXIDOREDUCTASE (PA1024) by JACOB BALL A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2018 Copyright by Jacob Aaron Ball 2018 MECHANISTIC STUDIES ON D-ARGININE DEHYDROGENASE AND FUNCTIONAL ANNOTATION OF A NOVEL NADH:QUINONE OXIDOREDUCTASE (PA1024) by JACOB BALL Committee Chair: Giovanni Gadda Committee: Dabney Dixon Donald Hamelberg Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University August 2018 iv DEDICATION This dissertation is dedicated to my parents Hollis and Cookie Ball. v ACKNOWLEDGEMENTS I would like to thank my family for supporting me through this journey. My parents, Hollis and Cookie, and siblings – brothers Butler, Adam, Jeremy, and sister, Carrie – all have been a crucial support this entire time. I would like to thank my advisor, Dr. Giovanni Gadda, for pushing me in the right direction to become the driven individual I am today. I am forever indebted to him for lending his expertise and knowledge. I never envisioned myself obtaining a PhD, but he helped me realize I did have the strength and ability to do so. I am grateful for my committee members. Dr. Dabney Dixon – thank you for asking tough questions and helping me improve my CV to a high standard. Dr. Donald Hamelberg – thank you for being an outstanding professor in physical chemistry and offering advice. I would like to thank lab members past and present – Swathi, Francesca, Crystal, Yvette, Rizuan, Elvira, Dan, Daniel, Quan, Archana, Renata, Hossein, Elias, Chris, and Maria – and many more. Swathi, you were an excellent mentor when I first joined the lab as an undergraduate. Finally, I would like to thank my girlfriend, Beibei, who improved many aspects of my life and gave me constant support and encouragement. Completion of this degree would not have been possible without you all. The work presented in this dissertation was supported by an MBD graduate fellowship. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ V LIST OF TABLES ....................................................................................................... XIII LIST OF FIGURES ..................................................................................................... XIV LIST OF SCHEMES ................................................................................................... XVI LIST OF ABBREVIATIONS ................................................................................... XVII 1 INTRODUCTION ...................................................................................................... 1 1.1 Substrate specificity of flavoenzymes that oxidize D-amino acids .................. 1 1.1.1 Abstract ............................................................................................................ 1 1.1.2 Introduction ..................................................................................................... 1 1.1.3 Importance of D-amino acids ......................................................................... 2 1.1.4 Flavoenzymes and Cα-N bond oxidation ........................................................ 3 1.1.5 D-amino acid oxidase: an enzyme with preference for neutral substrates ... 9 1.1.6 D-arginine dehydrogenase: an enzyme with preference for cationic substrates 15 1.1.7 D-aspartate oxidase: an enzyme with preference for anionic substrates.... 16 1.1.8 Active site lid.................................................................................................. 18 1.1.9 General principles about structure and specificity ...................................... 20 1.1.10 Closing thoughts and future directions ...................................................... 21 1.1.11 References ................................................................................................... 23 vii 1.2 Quinone detoxification by flavoenzymes ......................................................... 37 1.2.1 Quinones........................................................................................................ 37 1.2.2 Two-electron reduction of quinones............................................................. 38 1.2.3 References ..................................................................................................... 39 1.3 Specific Goals ..................................................................................................... 40 2 AMINE OXIDATION BY D-ARGININE DEHYDROGENASE IN PSEUDOMONAS AERUGINOSA ............................................................................................. 42 2.1 Abstract .............................................................................................................. 42 2.2 Introduction ....................................................................................................... 42 2.3 D-amino acids and their metabolism ............................................................... 44 2.4 Structural features of PaDADH ....................................................................... 46 2.4.1 Overall fold of PaDADH............................................................................... 46 2.4.2 FAD-binding domain .................................................................................... 47 2.4.3 Substrate binding-domain ............................................................................ 49 2.4.4 Structural Comparison of PaDADH with D-amino Acid Oxidase (DAAO)49 2.5 Substrate specificity of PaDADH ..................................................................... 51 2.5.1 E87 dictates substrate specificity .................................................................. 53 2.5.2 A hydrophobic cage and a flexible loop extends substrate scope ................ 54 2.5.3 Substrate
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