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Copyright by Christopher James Thibodeaux 2010 Copyright by Christopher James Thibodeaux 2010 The Dissertation Committee for Christopher James Thibodeaux Certifies that this is the approved version of the following dissertation: Mechanistic Studies of Two Enzymes that Employ Common Coenzymes in Uncommon Ways Committee: Hung-wen Liu, Supervisor Eric Anslyn Walter Fast Kenneth A. Johnson Christian P. Whitman Mechanistic Studies of Two Enzymes that Employ Common Coenzymes in Uncommon Ways by Christopher James Thibodeaux, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August, 2010 Dedication To all whom have made significant contributions to my life: I am eternally grateful. Acknowledgements First and foremost, I would like to thank Dr. Liu for providing me with the opportunity to work at the cutting edge of biochemical research, and for allowing me the freedom to explore and develop my scientific interests. In addition, I would like to thank the numerous other members of the Liu group (both past and present) for their helpful insights, stimulating conversations, and jovial personalities. They have all helped to immeasurably enrich my experience as a graduate student, and I would consider myself lucky to ever have another group of coworkers as friendly and as helpful as they all have been. Special thanks need to be attributed to Drs. Mark Ruszczycky, Chad Melançon, Yasushi Ogasawara, and Svetlana Borisova for their helpful suggestions and discussions at various points throughout my research, to Dr. Steven Mansoorabadi for performing the DFT calculations and for the many interesting conversations we have had over the years, and to Mr. Wei-chen Chang for continually supplying me with a variety of useful compounds that have been instrumental in the completion of this work. I would also like to thank Dr. Yung-nan Liu, Miss Sun He, and Miss Meilan Wu for assisting me with some of the molecular cloning and protein purification, Dr. Zhihua Tao for constructing several of the ACC deaminase mutants, and to Steve Sorey in the NMR facility for his assistance in collecting some of the NMR data. v Finally, none of this would have been possible (or worthwhile) without the support of my family, especially my wife and parents. vi Mechanistic Studies of Two Enzymes that Employ Common Coenzymes in Uncommon Ways Christopher James Thibodeaux, Ph.D. The University of Texas at Austin, 2010 Supervisor: Hung-wen Liu Enzymes are biological catalysts which greatly accelerate the rates of chemical reactions, oftentimes by many orders of magnitude over the uncatalyzed reaction. The remarkable catalytic rate enhancement afforded by enzymes derives ultimately from the structure and chemical properties of the enzyme active sites, which allow enzymes to selectively bind to their substrates and to stabilize high energy chemical species and unstable intermediates along the reaction coordinate. To enhance their catalytic ability, many enzymes have also evolved to require coenzymes for optimal activity. These coenzymes often provide chemical functionality and reactivity that are not accessible by the twenty canonical amino acids and, hence, coenzymes serve to greatly enhance the diversity of chemical reactions that can be mediated by enzymes. The work described in this dissertation focuses on mechanistic studies of two enzymes that use common coenzymes in unusual ways. In the first section of this work, studies will focus on the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2), an essential enzyme in isoprenoid biosynthesis that employs a flavin mononucleotide (FMN) vii coenzyme for catalysis. In most biological systems, flavin coenzymes mediate electron transfer reactions. However, the IDI-2 catalyzed reaction involves no net redox change, raising questions as to the role of the flavin in the chemical mechanism. The chemical mechanism of IDI-2 will be interrogated with a combination of spectroscopic studies and biochemical techniques. Our studies suggest that the flavin coenzyme of IDI-2 may employ a novel mode of flavin-dependent catalysis involving acid/base chemistry. In the second section of this dissertation, attention will be focused on elucidating the chemical mechanism of 1-aminocyclopropane-1-carboxylate deaminase (ACCD), an enzyme that plays a role in regulating the production of the potent plant hormone, ethylene. ACCD is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes a C-C bond cleavage event that is unique among the catalytic cycles of PLP-dependent enzymes. Altogether, our mechanistic studies of IDI-2 and ACCD help to illustrate the catalytic diversity of common coenzymes, and demonstrate that some enzymes have evolved to exploit readily available coenzymes for atypical reactions. viii Table of Contents MECHANISTIC STUDIES OF THE TYPE II ISOPENTENYL DIPHOSPHATE:DIMETHYLALLY DIPHOSPHATE ISOMERASE (IDI-2) FROM STAPHYLOCOCCUS AUREUS 1 Chapter 1 The Essential Role of Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerases in Isoprenoid Biosynthesis ...........................................................1 1.1 Background ..............................................................................................1 1.2 Biosynthesis of Isprenoid Units................................................................1 1.3 Chain Elongation in Isoprenoid Biosynthesis...........................................5 1.4 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase (IDI)....8 1.5 Mechanistic Studies of IDI-1....................................................................8 1.6 Identification and Initial Characterization of IDI-2 ...............................15 1.7 Flavoenzymes that Catalyze Reactions with no Net Redox Change .....19 1.8 Mechanistic Considerations for IDI-2 ...................................................25 1.9 Thesis Statement .....................................................................................29 Chapter 2 Spectroscopic, Biochemical, and Computational Studies of the IDI-2 Bound Flavin Intermediate ...........................................................................31 2.1 Introduction.............................................................................................31 2.2 Methods...................................................................................................32 2.2.1 Site-directed mutagenesis of the wt idi2 gene from Staphylococcus aureus...................................................................................................32 2.2.2 Transformation of E. coli M15[pRep4] cells with IDI-2/pQES constructs ........................................................................32 2.2.3 Overexpression of IDI-2 and its mutants....................................34 2.2.4 Purification of IDI-2 and its mutants ..........................................34 ix 2.2.5 UV-visible absorption spectroscopy of wt IDI-2........................35 2.2.6 EPR analysis of IDI-2 bound flavin semiquinone ......................36 2.2.7 Comparison of flavin intermediate absorption at pH 8.0 to free FMNH2 absorption at pH 4.0....................................................37 2.2.8 pH dependence of flavin intermediate absorption ......................38 2.2.9 UV-visible absorption spectroscopy of IDI-2 mutant enzymes................................................................................................38 2.2.10 DFT calculations.......................................................................38 2.3 Results and Discussion ...........................................................................39 2.3.1 Characterization of the IDI-2 bound semiquinone......................39 2.3.2 A flavin intermediate forms upon IPP binding...........................43 2.3.3 Spectroscopic comparison of the IDI-2 bound flavin intermediate to free FMN..........................................................................................45 2.3.4 pH dependence of flavin intermediate absorption ......................49 2.3.5 Absorption properties of IDI-2 mutant enzymes ........................50 2.3.6 DFT calculations provide insight into the factors that govern the IDI-2 flavin intermediate absorption .............................................54 2.4 Conclusions, Mechanistic Implications, and Future Directions .............59 2.4.1 The flavin intermediate is most likely an FMNH- species..........59 2.4.2 The protonation state of the reduced, IDI-2 bound flavin has mechanistic relevance....................................................................61 2.4.3 Considering an isomerization mechanism involving single electron transfer.........................................................................63 Chapter 3 Defining the Biochemical Properties of the IDI-2 Catalyzed Reaction .......................................................................................................68 3.1 Introduction.............................................................................................68 3.2 Methods...................................................................................................69 3.2.1 General........................................................................................69 x 3.2.2 Determination of steady state kinetic parameters .......................70 3.2.3 Determination of the equilibrium constant .................................71 3.2.4 pH dependence of the steady state kinetic
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