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On the Biochemistry, Mechanism and Physiological Role of Fungal Nitronate Monooxygenase

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On the Biochemistry, Mechanism and Physiological Role of Fungal Nitronate Monooxygenase Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry Spring 4-27-2011 On the Biochemistry, Mechanism and Physiological Role of Fungal Nitronate Monooxygenase Kevin Francis Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Part of the Chemistry Commons Recommended Citation Francis, Kevin, "On the Biochemistry, Mechanism and Physiological Role of Fungal Nitronate Monooxygenase." Dissertation, Georgia State University, 2011. https://scholarworks.gsu.edu/chemistry_diss/51 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]. ON THE BIOCHEMISTRY, MECHANISM AND PHYSIOLOGICAL ROLE OF FUNGAL NITRONATE MONOOXYGENASE by KEVIN FRANCIS Under the Direction of Giovanni Gadda ABSTRACT Nitronate monooxygenase (E.C. 1.13.11.16), formerly known as 2-nitropropane dioxygenase (EC 1.13.11.32), is a flavin dependent enzyme that catalyzes the oxidation of nitronates to their corresponding carbonyl compounds and nitrite. Despite the fact that the enzyme was first isolated from Neurospora crassa 60 years ago, the biochemical and physiological properties of nitronate monooxygenase have remained largely elusive. This dissertation will present the work that established both the catalytic mechanism and physiological role of the fungal enzyme. The biological and biochemical properties of propionate-3-nitronate, the recently discovered physiological substrate for nitronate monooxygenase, will be extensively reviewed. The nitronate is produced by a variety of variety leguminous plants and fungi and is a potent and irreversible inhibitor of succinate dehydrogenase. Nitronate monooxygenase allows N. crassa to overcome the toxicity of propionate-3-nitronate as demonstrated by in vivo studies of the yeast, which showed that the wild-type can grow in the presence of the toxin whereas a knock out mutant that lacks the gene encoding for the enzyme could not. In addition to establishing the physiological role of nitronate monooxygenase, the work presented here demonstrates that the catalytic mechanism of the enzyme involves the formation of an anionic flavosemiquinone intermediate. This intermediate is stabilized by the protonated form of an active site histidine residue (His-196) that acts as an electrostatic catalyst for the reaction as demonstrated by pH studies of the reductive half reaction of the enzyme. Histidine 196 also serves as the catalytic base for the reaction of the enzyme with nitroethane as substrate as revealed through mutagenesis studies in which the residue was replaced with an asparagine. The kinetic implications of branching of reaction intermediates in enzymatic catalysis are also demonstrated through studies of the kinetic isotope effects of nitronate monooxygenase with 2 1,1-[ H2]-nitroethane as substrate. Finally the use of competitive inhibitors as a probe of enzyme structure will be presented through a study of the inhibition of nitronate monooxygenase with mono-valent inorganic ions. The dissertation will close with unpublished work on the enzyme and concluding remarks concerning the biochemistry and physiology of nitronate monooxygenase. INDEX WORDS: Nitronate monooxygenase, 2-Nitropropane dioxygenase, Kinetic isotope and pH effects, Flavin semiquinone, Nitroalkanes, 3-Nitropropionate, Propionate-3-nitronate ON THE BIOCHEMISTRY, MECHANISM AND PHYSIOLOGICAL ROLE OF FUNGAL NITRONATE MONOOXYGENASE by KEVIN FRANCIS 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 2011 Copyright by Kevin Francis 2011 ON THE BIOCHEMISTRY, MECHANISM AND PHYSIOLOGICAL ROLE OF FUNGAL NITRONATE MONOOXYGENASE by KEVIN FRANCIS Committee Chair: Giovanni Gadda Committee: Dabney White Dixon Aimin Liu Binghe Wang Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2011 iv DEDICATION This dissertation is lovingly dedicated to Mary Arliss Quinlan Couey who was tragically killed by a drunk driver on March 31, 2009. You are forever missed by many. v ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Giovanni Gadda for his continued support and advice throughout my PhD career. In addition, I would also like to thank my committee members and all lab mates past and present. It could not have been done without you all. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ......................................................................................................... v 1 CHAPTER I ................................................................................................................ 1 GENERAL INTRODUCTION: NITRONATE MONOOXYGENASE AND OXIDATION OF NITRONATES BY FLAVIN DEPENDANT ENZYMES .................................................. 1 1.1 Abstract ................................................................................................................ 1 1.2 Isolation and Initial Characterization of Nitronate Monooxygenase as a 2- Nitropropane Oxidizing Enzyme ............................................................................. 1 1.3 Classification of the Fungal Enzyme as Nitronate Monooxygenase ............... 4 1.4 General Mechanisms of Flavin Dependent Nitroalkane Oxidizing Enzymes 7 1.5 Specific Goals Addressed in the Dissertation ................................................. 10 1.6 References .......................................................................................................... 12 2 CHAPTER II ............................................................................................................ 18 THE BIOLOGY AND BIOCHEMISTRY OF 3-NITROPROPIONATE: A POTENT INHIBITOR OF SUCCINATE DEHYDROGENASE............................................................ 18 2.1 Abstract .............................................................................................................. 18 2.2 Introduction ....................................................................................................... 18 2.3 Chemical Properties of 3-Nitropropionate ..................................................... 19 2.4 Biological Occurrence and Function of 3-Nitropropionate ........................... 20 2.5 Biosynthesis of 3-Nitropropionate ................................................................... 23 2.6 Biochemical Basis for 3-Nitropropionate Toxicity ......................................... 25 2.7 Physiological Effects of 3-Nitropropionate Poisoning.................................... 30 2.8 3-Nitropropionate Oxidizing Enzymes ............................................................ 31 vii 2.9 Physiological Role of 3-Nitropropionate Oxidizing Enzymes ....................... 35 2.10 References ..................................................................................................... 37 3 CHAPTER III ........................................................................................................... 43 INVOLVEMENT OF A FLAVOSEMIQUINONE IN THE ENZYMATIC OXIDATION OF NITROALKANES CATALYZED BY 2-NITROPROPANE DIOXYGENASE ........... 43 3.1 Summary ............................................................................................................ 43 3.2 Introduction ....................................................................................................... 44 3.3 Experimental Procedures ................................................................................. 46 3.4 Results ................................................................................................................ 53 3.5 Discussion ........................................................................................................... 66 3.6 Acknowledgements ............................................................................................ 74 3.7 References .......................................................................................................... 76 4 CHAPTER IV ........................................................................................................... 83 PROBING THE CHEMICAL STEPS OF NITROALKANE OXIDATION CATALYZED BY 2-NITROPROPANE DIOXYGENASE WITH SOLVENT VISCOSITY, PH, AND SUBSTRATE KINETIC ISOTOPE EFFECTS ....................................................................... 83 4.1 Abstract .............................................................................................................. 83 4.2 Introduction ....................................................................................................... 84 4.3 Materials and Methods ..................................................................................... 87 4.4 Results ................................................................................................................ 91 4.5 Discussion ........................................................................................................... 99 4.6 Acknowledgments............................................................................................ 108 4.7 Supporting Information .................................................................................. 109 viii 4.8 References .......................................................................................................
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