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Kinetic and Structural Studies on Flavin-Dependent Enzymes Involved in Glycine Betaine Biosynthesis and Propionate 3-Nitronate Detoxification

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Kinetic and Structural Studies on Flavin-Dependent Enzymes Involved in Glycine Betaine Biosynthesis and Propionate 3-Nitronate Detoxification Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry Spring 5-11-2015 Kinetic and Structural Studies on Flavin-dependent Enzymes involved in Glycine Betaine Biosynthesis and Propionate 3-nitronate Detoxification Francesca Salvi Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Recommended Citation Salvi, Francesca, "Kinetic and Structural Studies on Flavin-dependent Enzymes involved in Glycine Betaine Biosynthesis and Propionate 3-nitronate Detoxification." Dissertation, Georgia State University, 2015. https://scholarworks.gsu.edu/chemistry_diss/106 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]. KINETIC AND STRUCTURAL STUDIES ON FLAVIN-DEPENDENT ENZYMES INVOLVED IN GLYCINE BETAINE BIOSYNTHESIS AND PROPIONATE 3-NITRONATE DETOXIFICATION by FRANCESCA SALVI Under the Direction of Giovanni Gadda, PhD ABSTRACT Flavin-dependent enzymes are characterized by an amazing chemical versatility and play important roles in different cellular pathways. The FAD-containing choline oxidase from Arthrobacter globiformis oxidizes choline to glycine betaine and retains the intermediate betaine aldehyde in the active site. The reduced FAD is oxidized by oxygen. Glycine betaine is an important osmoprotectant accumulated by bacteria, plants, and animals in response to stress conditions. The FMN-containing nitronate monooxygenase detoxifies the deadly toxin propionate 3-nitronate which is produced by plants and fungi as defense mechanism against herbivores. The catalytic mechanism of fungal nitronate monooxygenase (NMO) was characterized, but little is known about bacterial NMOs. In this dissertation the crystal structure of choline oxidase in complex with glycine betaine was solved and the roles of the residue F357 in the oxidative half reaction investigated by combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects and molecular dynamics simulations. Expression trials of human choline dehydrogenase were carried out and a homology model based on choline oxidase was generated. A bacterial nitronate monooxygenase from Pseudomonas aeruginosa PAO1 Pa-NMO was kinetically and structurally characterized and four conserved motifs were described that identify Class 1 NMO. Two hypothetical NMOs from P. aeruginosa PAO1 and one from Helicobacter pylori not carrying the motifs of Class 1 NMO were cloned and tested for nitronate monooxygenase activity. The crystal structure of choline oxidase in complex with glycine betaine highlighted two different conformations of loop 250-255 at the dimer interface that is proposed to control substrate access to the active site. The side chain of F357 was associated with a slow isomerization in the oxidation of the reduced FAD of choline oxidase. The first crystal structure of an NMO highlighted active site residues for site-directed mutagenesis studies. The gene function prediction for NMO was improved with the four conserved motifs of Pa-NMO. The two hypothetical NMOs from P. aeruginosa PAO1 were shown to possess a different enzymatic activity and to identify two distinct classes of enzymes. The hypothetical NMO from H. pilory did not exhibit NMO activity and contained iron as cofactor. INDEX WORDS: Flavin, Choline oxidase, Nitronate monooxygenase, Propionate 3-nitronate, Choline dehydrogenase, Gene function prediction KINETIC AND STRUCTURAL STUDIES ON FLAVIN-DEPENDENT ENZYMES INVOLVED IN GLYCINE BETAINE BIOSYNTHESIS AND PROPIONATE 3-NITRONATE DETOXIFICATION by FRANCESCA SALVI 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 2015 Copyright by Francesca Salvi 2015 KINETIC AND STRUCTURAL STUDIES ON FLAVIN-DEPENDENT ENZYMES INVOLVED IN GLYCINE BETAINE BIOSYNTHESIS AND PROPIONATE 3-NITRONATE DETOXIFICATION by FRANCESCA SALVI Committee Chair: Giovanni Gadda Committee: Irene T. Weber Donald Hamelberg Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2015 iv DEDICATION To my husband Roberto Orrù . v ACKNOWLEDGEMENTS I thank my supervisor and my committee members for their support, all my lab members and collaborators for the time spent together. The work of this dissertation was supported by an MBDAF fellowship. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ......................................................................................................v LIST OF TABLES ................................................................................................................. xii LIST OF FIGURES ...............................................................................................................xiv LIST OF SCHEMES ..............................................................................................................xix 1 CHAPTER 1: INTRODUCTION ......................................................................................1 1.1 General features of flavin-dependent enzymes ................................................1 1.2 Structural features of selected flavin-dependent enzymes ............................ 10 1.3 Oxidases, monooxygenases, and dehydrogenases .......................................... 17 1.4 Glycine betaine biosynthesis .......................................................................... 32 1.5 Nitronate monooxygenases and the detoxification of propionate 3-nitronate . ......................................................................................................................... 41 1.6 Specific goals ................................................................................................... 52 1.7 References ....................................................................................................... 56 2 CHAPTER II: CRYSTAL STRUCTURE OF CHOLINE OXIDASE IN COMPLEX WITH THE REACTION PRODUCT GLYCINE BETAINE ............................................... 74 2.1 Abstract .......................................................................................................... 74 2.2 Introduction .................................................................................................... 74 2.3 Materials and methods ................................................................................... 77 2.4 Results ............................................................................................................. 79 vii 2.5 Discussion ....................................................................................................... 87 2.6 Conclusion ...................................................................................................... 92 2.7 Acknowledgements ......................................................................................... 93 2.8 References ....................................................................................................... 93 3 CHAPTER III: INVOLVEMENT OF F357 IN AN ISOMERIZATION PARTIALLY RATE LIMITING FOR FLAVIN OXIDATION IN CHOLINE OXIDASE........................ 98 3.1 Abstract .......................................................................................................... 98 3.2 Introduction .................................................................................................... 99 3.3 Materials and methods ................................................................................. 102 3.4 Results ........................................................................................................... 107 3.5 Discussion ..................................................................................................... 121 3.6 Conclusion .................................................................................................... 127 3.7 References ..................................................................................................... 129 4 CHAPTER IV: HUMAN CHOLINE DEHYDROGENASE: MEDICAL PROMISES AND BIOCHEMICAL CHALLENGES .............................................................................. 131 4.1 Abbreviations ............................................................................................... 131 4.2 Abstract ........................................................................................................ 131 4.3 Introduction .................................................................................................. 132 4.4 Physiological roles of human CHD .............................................................. 135 4.5 Medical relevance of CHD ........................................................................... 137 viii 4.6 Subcellular localization of mammalian CHD .............................................. 140 4.7 Glucose-Methanol-Choline enzyme oxidoreductase superfamily ............... 141 4.8 Purification of CHD ..................................................................................... 143 4.9 Biochemical properties of CHD ................................................................... 145 4.10 Homology model of human CHD ................................................................. 148 4.11 Comparison of CHD
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