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Roles of Serine 101, Histidine 310 and Valine 464 in the Reaction Catalyzed by Choline Oxidase from Arthrobacter Globiformis

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Roles of Serine 101, Histidine 310 and Valine 464 in the Reaction Catalyzed by Choline Oxidase from Arthrobacter Globiformis Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry 3-5-2010 Roles of Serine 101, Histidine 310 and Valine 464 in the Reaction Catalyzed by Choline Oxidase from Arthrobacter Globiformis Steffan Finnegan Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Part of the Chemistry Commons Recommended Citation Finnegan, Steffan, "Roles of Serine 101, Histidine 310 and Valine 464 in the Reaction Catalyzed by Choline Oxidase from Arthrobacter Globiformis." Dissertation, Georgia State University, 2010. https://scholarworks.gsu.edu/chemistry_diss/37 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]. ROLES OF SERINE 101, HISTIDINE 310 AND VALINE 464 IN THE REACTION CATALYZED BY CHOLINE OXIDASE FROM ARTHROBACTER GLOBIFORMIS by STEFFAN FINNEGAN Under the Direction of Giovanni Gadda ABSTRACT The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions, as such it is of potential interest for the genetic engineering of crops that do not naturally possess efficient pathways for the synthesis of glycine betaine, and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, one of the best characterized enzymes belonging to this pathway is the flavin-dependent choline oxidase from Arthrobacter globiformis. In this enzyme, choline oxidation proceeds through two reductive half- reactions and two oxidative half-reactions. In each of the reductive half-reactions the FAD cofactor is reduced to the anionic hydroquinone form (2 e- reduced) which is followed by an oxidative half-reaction where the reduced FAD cofactor is reoxidized by molecular oxygen with formation and release of hydrogen peroxide. In this dissertation the roles of selected residues, namely histidine at position 310, valine at position 464 and serine at position 101, that do not directly participate in catalysis in the reaction catalyzed by choline oxidase have been elucidated. The effects on the overall reaction kinetics of these residues in the protein matrix were investigated by a combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects as well as X-ray crystallography. A comparison of the kinetic data obtained for the variant enzymes to previous data obtained for wild-type choline oxidase are consistent with the valine residue at position 464 being important for the oxidative half-reaction as well as the positioning of the catalytic groups in the active site of the enzyme. The kinetic data obtained for the serine at position 101 shows that serine 101 is important for both the reductive and oxidative half-reactions. Finally, the kinetic data for histidine at position 310 suggest that this residue is essential for both the reductive and oxidative half-reactions. INDEX WORDS: Choline oxidase, Flavin oxidation, Oxygen reactivity, Proton-transfer network, Chemical mechanism, Flavoprotein. ROLES OF SERINE 101, HISTIDINE 310 AND VALINE 464 IN THE REACTION CATALYZED BY CHOLINE OXIDASE FROM ARTHROBACTER GLOBIFORMIS by STEFFAN FINNEGAN 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 2010 Copyright by Steffan Finnegan 2010 ROLES OF SERINE 101, HISTIDINE 310 AND VALINE 464 IN THE REACTION CATALYZED BY CHOLINE OXIDASE FROM ARTHROBACTER GLOBIFORMIS by STEFFAN FINNEGAN Committee Chair: Dr. Giovanni Gadda Committee: Dr. Markus W. Germann Dr. Aimin Liu Electronic Version Approved by: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2010 v TABLE OF CONTENTS LIST OF TABLES ix LIST OF FIGURES xi LIST OF SCHEMES xv CHAPTER I 1 INTRODUCTION 1 1.1 Flavin dependent enzymes ............................................................................................. 1 1.2 Selected structural features of flavoenzymes with different oxygen reactivity ........ 7 1.3 Oxygen reactivity.......................................................................................................... 12 1.3.1. General oxygen chemistry .................................................................................... 12 1.3.2. Enzymes and oxygen ............................................................................................. 15 1.4. Choline Oxidase from Arthrobacter globiformis ........................................................ 28 1.5. Specific Goals ................................................................................................................ 32 1.6. References ..................................................................................................................... 35 CHAPTER II 57 CRYSTALLOGRAPHIC, SPECTROSCOPIC, AND COMPUTATIONAL ANALYSIS OF A FLAVIN C4A-OXYGEN ADDUCT IN CHOLINE OXIDASE. 57 2.1. Abstract ......................................................................................................................... 57 2.2. Introduction .................................................................................................................. 57 2.3. Materials and methods................................................................................................. 61 2.4. Results and discussion .................................................................................................. 65 2.5. Acknowledgment .......................................................................................................... 79 2.6. References ..................................................................................................................... 80 vi CHAPTER III 87 SUBSTITUTION OF AN ACTIVE SITE VALINE UNCOVERS A KINETICALLY SLOW EQUILIBRIUM BETWEEN COMPETENT AND INCOMPETENT FORMS OF CHOLINE OXIDASE 87 3.1. Abbreviations ................................................................................................................ 87 3.2. Abstract ......................................................................................................................... 87 3.3. Introduction .................................................................................................................. 88 3.4. Experimental procedures ............................................................................................ 94 3.5. Results ........................................................................................................................... 97 3.6. Discussion .................................................................................................................... 111 3.7. Appendix ..................................................................................................................... 117 3.8. Acknowledgements ..................................................................................................... 121 3.9. References ................................................................................................................... 121 CHAPTER IV 130 ROLE OF VAL464 IN THE FLAVIN OXIDATION REACTION CATALYZED BY CHOLINE OXIDASE 130 4.1. Abbreviations .............................................................................................................. 130 4.1. Abstract ....................................................................................................................... 130 4.2. Introduction ................................................................................................................ 131 4.3. Experimental procedures .......................................................................................... 135 4.4. Results ......................................................................................................................... 140 4.5. Discussion .................................................................................................................... 151 4.6. Acknowledgements ..................................................................................................... 156 vii 4.7. References ................................................................................................................... 157 CHAPTER V 163 ON THE IMPORTANCE OF SER101 FOR OVERALL TURNOVER IN CHOLINE OXIDASE 163 5.1. Abstract ....................................................................................................................... 163 5.2. Introduction ................................................................................................................ 163 5.3. Experimental Procedures .......................................................................................... 165 5.4. Results ......................................................................................................................... 168 5.5. Discussion .................................................................................................................... 174 5.6. Acknowledgement ...................................................................................................... 176 5.7. References ..................................................................................................................
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