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MARTI-ARBONA-DISSERTATION.Pdf (5.556Mb) MECHANISTIC CHARACTERIZATION OF MEMBERS OF THE AMIDOHYDROLASE SUPERFAMILY A Dissertation by RICARDO MARTÍ ARBONA 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 2006 Major Subject: Chemistry MECHANISTIC CHARACTERIZATION OF MEMBERS OF THE AMIDOHYDROLASE SUPERFAMILY A Dissertation by RICARDO MARTÍ ARBONA 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 Approved by: Chair of Committee, Frank M. Raushel Committee Members, Paul A. Lindahl Eric E. Simanek Jerry Tsai Head of Department, Emile A. Schweikert December 2006 Major Subject: Chemistry iii ABSTRACT Mechanistic Characterization of Members of the Amidohydrolase Superfamily. (December 2006) Ricardo Martí Arbona, B.S., Universidad de Puerto Rico, at Río Piedras Chair of Advisory Committee: Dr. Frank M. Raushel The amidohydrolase superfamily is a functionally diverse group of enzymes found in every organism sequenced to date. The landmark for this superfamily is the conservation of a (/)8-barrel structural fold. Isoaspartyl dipeptidase (IAD) from Escherichia coli catalyzes the hydrolytic cleavage of -aspartyl dipeptides. Structural studies of the wild-type enzyme demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for all four metal-substituted forms of the wild-type enzyme and the site-directed mutants, E77Q and Y137F. Structural analysis of IAD with the bound substrate and site-directed mutagenesis shows the importance of the side chains of residues Glu-77, Tyr-137, Arg-169, Arg-233, Asp- 285, and Ser-289 in the substrate binding and hydrolysis. The reaction mechanism for the hydrolysis of dipeptides by IAD is initiated by the polarization of the amide bond via complexation to the -metal and the hydrogen bond to Tyr-137. Asp-385 participates in the activation of the bridging hydroxide for nucleophilic attack at the peptide carbon center. The lately protonated Asp-285 donates the proton to the -amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond. N-formimino-L-glutamate iminohydrolase (HutF) from iv Pseudomonas aeruginosa acts in the deimination of the fourth intermediate of the histidine degradation pathway, N-formimino-L-glutamate. An amino acid sequence alignment between HutF and other members of the amidohydrolase superfamily containing mononuclear metal centers suggests that the residues Glu-235, His-269, and Asp-320 are involved in substrate binding and deimination. Site-directed mutagenesis of Glu-235, His-269, and Asp-320, in conjunction with the analysis of the four metal- substituted enzyme forms and pH-rate profiles provides valuable information toward the proposal of a mechanism for deimination of N-formimino-L-glutamate by HutF. This information suggests that the reaction is initiated by the activation of the hydrolytic water through base catalysis via His-269. The enhanced nucleophile attacks the formimino carbon center. In a concerted reaction, Asp-320 deprotonates the hydroxide nucleophile, and His-269 donates a proton to the terminal amino of the iminium group resulting in the collapse of the tetrahedral intermediate, the cleavage of the carbon-nitrogen bond and the release of the products. v DEDICATION To my wife, Denisse, my complement, inspiration and support, who makes me whole, and brings equilibrium and control to this chaos that people call life; to my parents, Angel and Josefina, who have been always there to laugh and cry with me, to share my achievements and disappointments, to listen, and help me through difficult times and to show me the way; to my brothers, Angel, Jonathan and Edgar, my best friends and supporters; and to my grandparents, Cheo and Pancha, who have been my biggest example of strength, devotion and accomplishment. vi ACKNOWLEDGMENTS I would like to thank Dr. Vicente Fresquet for everything he taught me and for his support throughout my graduate studies. Also, I thank my advisor, Dr. Frank M. Raushel, for his guidance and support and my committee members: Dr. Paul A. Lindahl, Dr. Erik E. Simanek and Dr. Jerry Tsai. Many thanks go to my past and present colleagues from Raushel’s laboratory for their friendship and assistance through the years, especially Richard Hall for all the help with the spelling corrections of this dissertation. vii TABLE OF CONTENTS Page ABSTRACT.…………………………………………………………....................... iii DEDICATION.……………………………………………………………………... v ACKNOWLEDGMENTS .………………………………………………………… vi TABLE OF CONTENTS ………………………………………………………….. vii LIST OF FIGURES ………………………………………………………………... ix LIST OF TABLES …………………………………………………………………. xi CHAPTER I INTRODUCTION: THE AMIDOHYDROLASE SUPERFAMILY, A PARADIGM OF CATALYTIC DIVERSITY…………………… 1 II HIGH-RESOLUTION X-RAY STRUCTURE OF ISOASPARTYL DIPEPTIDASE FROM ESCHERICHIA COLI …………………….. 18 Materials and Methods …………………………………….. 22 Results.................................................................................... 25 Discussion ............................................................................. 32 III MECHANISM OF THE REACTION CATALYZED BY ISOASPARTYL DIPEPTIDASE FROM ESCHERICHIA COLI.…. 35 Materials and Methods …………………………………….. 39 Results.................................................................................... 48 Discussion ............................................................................. 55 IV FUNCTIONAL SIGNIFICANCE OF GLU-77 AND TYR-137 WITHIN THE ACTIVE SITE OF ISOASPARTYL DIPEPTIDASE …………………………………………………….. 63 Materials and Methods …………………………………….. 67 Results.................................................................................... 69 Discussion ............................................................................. 76 viii TABLE OF CONTENTS (continued) Page CHAPTER V ANNOTATING ENZYMES OF UNKNOWN FUNCTION: N- FORMIMINO-L-GLUTAMATE DEIMINASE IS A MEMBER OF THE AMIDOHYDROLASE SUPERFAMILY ……………….. 81 Materials and Methods …………………………………….. 85 Results.................................................................................... 94 Discussion.............................................................................. 100 VI MECHANISTIC CHARACTERIZATION OF N-FORMIMINO-L- GLUTAMATE IMINOHYDROLASE FROM PSEUDOMONAS AERUGINOSA ……………………………………………………... 111 Materials and Methods .......................................................... 114 Results.................................................................................... 118 Discussion.............................................................................. 123 VII SUMMARY AND CONCLUSIONS ……………………………… 128 REFERENCES ……………………………………………………………………. 140 VITA ……………………………………………………………………………….. 153 ix LIST OF FIGURES Page Figure 1.1: Schematic comparison of variations of the mononuclear or binuclear metal centers of enzymes within the amidohydrolase superfamily ………………………………………………………….. 5 Figure 2.1: X-ray crystal structure ribbon representation of a single subunit of IAD from Escherichia coli...................…………………….............. 26 Figure 2.2: Octamer quaternary structure of IAD.…………………………………. 27 Figure 2.3: Close-up view of the binuclear metal center of IAD………………….. 28 Figure 2.4: Close-up view of the active site of IAD with the complexed aspartate.. 30 Figure 2.5: Possible model for the binding of -Asp-Leu binding in the active site of IAD……………………………………………………… 31 Figure 2.6: Superposition of the active site region for the resting forms of the wild-type IAD, DHO, and PTE…..……………………………….. 33 Figure 3.1: X-ray crystal structure of IAD from Escherichia coli…………...…… 37 Figure 3.2: Close-up view of the active site of IAD with the complexed aspartate.. 39 Figure 3.3: pH-rate profiles of kcat and kcat/Km for different metal reconstituted IAD WT ( Zn, Co, Ni and Cd)………………………………... 52 Figure 3.4: Close-up view and potential hydrogen bonding interactions between -Asp-His and the D285N mutant…………………………… 55 Figure 4.1: Close-up view of the active site of the D285N mutant with bound -Asp-His, highlighted in gold bonds…………………………………. 66 Figure 4.2: The pH-rate profiles of kcat and kcat/Km for type (), E77Q () and Y137F mutant enzyme ()..…………..…………………………. 73 x LIST OF FIGURES (continued) Page Figure 4.3: Superposition of the active site region for the resting forms of wild-type IAD, the mutant E77Q, and the substrate bound complex of the mutant D285N………..……………………………….. 75 Figure 4.4: Superposition of the wild type IAD and the mutant Y137F…………... 76 Figure 5.1: Dendrogram of the Pa5106 homologues obtained from the microbial genomes……………………………………………………. 102 Figure 5.2: Amino acid sequence alignment of Pa5106, atrazine chlorohydrolase from Pseudomonas sp. and Tm0936 from Thermotoga maritima using CLUSTALW………………………………………………………...…… 105 Figure 5.3: A possible model for N-formimino-L-glutamate binding to the active site of Pa5106.……………………………………… ………………... 108 Figure 6.1: pH-rate profiles of kcat and kcat/Km for different metal reconstituted wild-type HutF( Zn, Cu, Ni and Cd)…………………………. 121 Figure 6.2: pH-rate profiles of kcat and kcat/Km for the mutant forms of HutF; E235A () and E235Q ()…………………………………………….. 122 xi LIST OF TABLES Page Table 3.1: Kinetic Parameters for IAD with Different Substrates..…………………. 50 Table 3.2: Kinetic Parameters for Different Metal Reconstituted IAD and Mutants.. 51 Table 3.3: Ionization Constants for Metal-Substituted
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