CORE Metadata, citation and similar papers at core.ac.uk Provided by Texas A&M University THE STRUCTURE AND MECHANISM OF BACTERIAL DIHYDROOROTASE A Dissertation by TAMIKO NEAL PORTER Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2004 Major Subject: Chemistry THE STRUCTURE AND MECHANISM OF BACTERIAL DIHYDROOROTASE A Dissertation by TAMIKO NEAL PORTER Submitted to Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved as to style and content by: ______________________________ ____________________________ Frank Raushel Gregory Reinhart (Chair of Committee) (Member) ______________________________ ____________________________ Victoria DeRose Paul Lindahl (Member) (Member) ______________________________ Emile Schweikert (Head of Department) December 2004 Major Subject: Chemistry iii ABSTRACT The Structure and Mechanism of Bacterial Dihydroorotase. (December 2004) Tamiko Neal Porter, B.S., Michigan State University Chair Advisory Committee: Dr. Frank Raushel Dihydroorotase (DHO) is a zinc metallo-enzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate and dihydroorotate. The X-ray crystal structure of the enzyme was obtained at a resolution of 1.7 Å. The pH-rate profiles for the hydrolysis of dihydroorotate or thio-dihydroorotate demonstrated that a single group of DHO must be unprotonated for maximal catalytic activity. The pH-rate profiles for the condensation of carbamoyl aspartate to dihydroorotate showed that a single group from the enzyme must be protonated for maximal catalytic activity. The native zinc ions within the active site of DHO were substituted with cobalt or CADmium by reconstitution of the apo-enzyme with divalent cations. The ionizations observed in the pH-rate profiles were dependent on the specific metal ion bound to the active site. Mutation of Asp-250 resulted in the loss of catalytic activity. These results are consistent with the formation of a hydroxide bridge between the two divalent cations that functions as the nucleophile during the hydrolysis of dihydroorotate. In addition, Asp- 250 is postulated to shuttle the proton from the bridging hydroxide to the leaving group amide during dihydroorotate hydrolysis. The X-ray crystal structure of DHO showed that the side-chain carboxylate of dihydroorotate is electrostatically interacting with Arg- 20, Asn-44 and His-254. Mutation of these residues resulted in the loss of catalytic activity, indicating that these residues are critical for substrate recognition. The thio- iv analog of dihydroorotate, (TDO) was found to be a substrate of DHO. A comprehensive chemical mechanism for DHO was proposed based on the experimental data presented in this dissertation. Armed with this understanding of the structure-function relationship of DHO, a rational approach was used to alter the substrate specificity of the enzyme. The R20/N44/H254 mutant of DHO was obtained and found to have increased activity on dihydrouracil compared to the wild-type enzyme. The sequence of the gene PA5541 from Pseudomonas aeruginosa has a glutamine at a position where most active DHO proteins have a histidine residue. Results from the characterization of PA5541 indicate that it is a functional DHO. v DEDICATION To my parents, Fred and Catherine Neal, who’ve always been my biggest cheerleaders; to my husband, Christopher, who is my best friend and supporter; and to our wonderful sons, Nickolas and Donovan. vi ACKNOWLEDGEMENTS I thank my adviser, Dr. Frank Raushel, for his guidance and support throughout my graduate career and my committee members: Dr. Gregory Reinhart, Dr. Pual Lindahl and Dr. Victoria DeRose. I would also like to thank Dr. Paul Fitzpatrick. Also, many thanks go to all of my past and present labmates for their friendship and assistance through the years. Finally, it is very important to thank Dr. Hazel Holden and Jim Thoden of the University of Wisconsin at Madison for their X-ray crystallography work. vii TABLE OF CONTENTS Page ABSTRACT .................................................................................................................iii DEDICATION...............................................................................................................v ACKNOWLEDGEMENTS ..........................................................................................vi LIST OF FIGURES ....................................................................................................viii LIST OF TABLES.........................................................................................................x CHAPTER I INTRODUCTION....................................................................................1 II THE X-RAY CRYSTAL STRUCTURE OF DIHYDROOROTASE FROM ESCHERICHIA COLI.................................................................17 Materials and Methods.......................................................................... 18 Results.................................................................................................. 19 Discussion ............................................................................................ 27 III MECHANISM OF THE DIHYDROOROTASE REACTION ................37 Materials and Methods.......................................................................... 41 Results.................................................................................................. 45 Discussion ............................................................................................ 55 IV THE ISOLATION OF A PROBABLE DIHYDROOROTASE FROM PSEUDOMONAS AERUGINOSA AND THE EVOLUTION OF DIHYDROOROTASE INTO A DIHYDROPYRIMIDINASE ...............62 Materials and Methods.......................................................................... 66 Results and Discussion ......................................................................... 70 V SUMMARY AND CONCLUSIONS ......................................................85 REFERENCES ............................................................................................................89 APPENDIX ............................................................................................................... 101 VITA......................................................................................................................... 116 viii LIST OF FIGURES Page Figure 1.1 Alignment of human and E. coli DHO sequences. ....................................... 5 Figure 1.2 Organization of the genes for CPS, DHO and ATC in various organisms. ... 7 Figure 2.1 Ribbon representation showing the distribution of secondary elements in the DHO dimer (PDB 1J79)………………..……………………………20 Figure 2.2 Representation of the TIM-barrel of the DHO monomer (PDB 1J79).. ...... 21 Figure 2.3 Overlay of the α-carbon backbone of the two subunits of DHO (PDB 1J79). .............................................................................................. 22 Figure 2.4 Overlay of the active sites of the two subunits of DHO (PDB 1J79). ......... 24 Figure 2.5 The active sites of DHO with bound carbamoyl aspartate (A) and dihydroorotate (B)..................................................................................... 25 Figure 2.6 Alignment of DHO sequences from human and E. coli.............................. 26 Figure 2.7 Mass spectra of native DHO (A) and the seleno-methionine derivative of DHO (B)............................................................................................... 28 Figure 2.8 Overlay of the active sites of DHO (blue), PTE (green) and Urease (red). 30 Figure 3.1 Representation of the binuclear metal center within the active site of DHO………………………………………………………………………..39 Figure 3.2 Time course for the hydrolysis of TDO at pH 8.0.. .................................... 47 Figure 3.3 pH dependence of the reactions catalyzed by Zn/Zn-DHO with carbamoyl aspartate (A, B), dihydroorotate (C, D) or thiodihydroorotate (E, F) as the varied substrate........................................ 50 Figure 3.4 Representation of the electrostatic interactions between the α-carboxylate of dihydroorotate and the side chains of Arg-20, Asn-44, and His-254.. ............................................................................... 53 Figure 3.5 Relative orientation of Asp-250 and the hydroxide that bridges the two divalent cations within the active site of dihydroorotase (PDB 1J79)......... 58 ix Page Figure 4.1 Alignment of P. aeruginosa DHO sequences, PA3527 and PA5541.......... 64 Figure 4.2 1.0 % Agarose gel demonstrating the amplification of PA5541. ................ 71 Figure 4.3 SDS-Page gel of the overexpression of PA5541. ....................................... 72 Figure 4.4 UV scan of the hydrolysis of thiodihydroorotate by PA5541. .................... 74 Figure 4.5 Substrate saturation curve for the hydrolysis of dihydroorotate by PA5541. ............................................................................................... 75 Figure 4.6 Substrate saturation curve for the synthesis of dihydroorotate by PA5541..................................................................................................... 76 Figure 4.7 12 % SDS-Page gel of the purification of the DHO mutant protein R20Q/N44D/H254Q.................................................................................. 79 x LIST OF TABLES Page