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STRUCTURE ACTIVITY RELATIONSHIPS IN PYRIDOXAL-5’-PHOSPHATE DEPENDENT ENZYMES by SANTIAGO LIMA (Under the Direction of Robert S. Phillips) ABSTRACT Pyridoxal-5’-phosphate (PLP) dependent enzymes are a large and catalytically diverse group of proteins primarily involved in the metabolism of amino acids, amino acid derived compounds, and amino sugars. In this work, three PLP-dependent enzymes, Homo sapiens kynureninase, Pseudomonas dacunhae L-aspartate-β-decarboxylase, and Pyrococcus furiosus tryptophan synthase β-subunit homolog are studied using a variety of biophysical methods. The novel crystal structures for these enzymes are presented, along with the structure of a kynureninase-inhibitor complex, and the analysis of a number of mutants generated to study specific structure activity relationships in them. The results of these analyses reveal the interactions that contribute to substrate specificity in kynureninase, a novel oligomerization scheme and catalytically important residues in aspartate-β-decarboxylase, and the elucidation of the kinetic properties of the P. furiosus tryptophan synthase β-subunit homolog. INDEX WORDS: pyridoxal-5’-phosphate, kynureninase, L-kynurenine hydrolase, 3- hydroxykynurenine, 2HZP, L-aspartate-beta-decarboxylase, aspartate-4- decarboxylase, tryptophan synthase beta subunit homolog, trpb2, tryptophan synthase STRUCTURE ACTIVITY RELATIONSHIPS IN PYRIDOXAL-5’-PHOSPHATE DEPENDENT ENZYMES by SANTIAGO LIMA BS, The University of Georgia, 2001 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2008 © 2008 Santiago Lima All Rights Reserved STRUCTURE ACTIVITY RELATIONSHIPS IN PYRIDOXAL-5’-PHOSPHATE DEPENDENT ENZYMES by SANTIAGO LIMA Major Professor: Robert S. Phillips Committee: Michael W. W. Adams Cory Momany Bi-Cheng Wang Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2008 iv DEDICATION I would like to dedicate this dissertation to my parents and sister who have stood by and supported, always believed in me, and encouraged me to believe in myself. I would also like to dedicate this work to my advisor and friend Dr. Robert S. Phillips for his invaluable guidance, patience, encouragement, and dedication to teaching. v ACKNOWLEDGEMENTS The work contained in this dissertation is the fruit of hard work, luck, and extensive collaboration. I would like to thank Dr. Cory Momany for supplying me with a great experience in learning the techniques and theory behind macromolecular X-ray crystallography. I would also like to thank Dr. Michael W.W. Adams, for his fair and cunning scientific input to my research, and Dr. Bi-Cheng Wang for taking time out of his busy schedule to serve on my committee. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES........................................................................................................................ vii LIST OF FIGURES ..................................................................................................................... viii INTRODUCTION AND LITERATURE REVIEW .......................................................................1 CHAPTER 1 Crystal Structure of Homo sapiens kynureninase........................................................18 2 Crystal Structure of Homo sapiens Kynureninase-3-hydroxy-hippuric Acid Inhibitor Complex; Insights into the Molecular Basis of Kynureninase Substrate Specificity ....................................................................................................57 3 The Crystal Structure of the Pseudomonas dacunhae L-Aspartate-Beta Decarboxylase Reveals a Novel Oligomeric Assembly for a Pyridoxal-5’-Phosphate Dependent Enzyme ......................................................................................................82 4 Atomic Structure and Substrate Specificity of the Pyrococcus furiosus Tryptophan Synthase β-Subunit Homolog ....................................................................................110 CONCLUSIONS..........................................................................................................................138 vii LIST OF TABLES Page Table 1.1: Summary of Crystallographic analysis.........................................................................47 Table 2.1: Summary of Crystallographic Analysis........................................................................78 Table 2.2: Summary of L-kynurenine and 3-OH-DL-kynuenine kinetic constants for wild type and kynureninase mutants...............................................................................................79 Table 3.1: Summary of Crystallographic analysis.......................................................................104 Table 4.1: Summary of Crystallographic analysis.......................................................................130 Table 4.1: Summary of kinetic constants measured for tryptophan synthases form S. typhimurium and P. furiosus. .............................................................................................................131 viii LIST OF FIGURES Page Figure 1.1: Ribbon drawing of the kynureninase biologically active unit with secondary structure elements colored in a blue to red progressing scheme. .................................................48 Figure 1.2: Stereo ribbon drawing of the kynureninase monomer with secondary structure elements colored in a blue to red progressing scheme ..................................................49 Figure 1.3: Map calculated with likelihood-weighted 2Fo-Fc coefficients for the area surrounding the pyridoxal-5’-phosphate-Lys-276 internal aldimine complex..............50 Figure 1.4: Stereo image of the kynureninase active site PLP hydrogen bonding interactions.....51 Figure 1.5: Structural superposition between dimers of human and Pseudomonas fluorescens kynureninases. ...............................................................................................................52 Figure 1.6: Stereo image of 3-hydroxy-L-kynurenine docked with human kynureninase. ...........53 Figure 1.7: Ribbon drawing of the Hkyn small domain S20-S21 β-hairpin and equivalent hairpins from the structures with PDB IDs 1qz9, 1m32, 1jf9, 1ibj, 1i29, 1h0c, 1fg3, 1d2f, 1bj4, and 2ch2......................................................................................................54 Figure 1.8: Comparison of the superimposed small domains of aspartate aminotransferase in open and closed conformation with the Homo sapiens kynureninase and the Pseudomonas fluorescens kynureninase .......................................................................55 Figure 1.9: Active site amino acid differences between human and P. fluorescens kynureninases in the vicinity of the 3-hydroxyl moiety of docked 3-hydroxy-L-kynurenine..............56 Figure 2.1: An electron density map showed continuous electron density for 3-HHA atoms ......80 ix Figure 2.2: Stereo representation of the interactions that stabilize 3-HHA (colored green, ball and stick atoms) within the Homo sapiens kynureninase active site ...................................81 Figure 3.1: ABDC monomer with successive secondary structure elements labeled..................105 Figure 3.2: The ABDC active site is composed of residues contributed from two monomer chains related by a 2-fold axis of symmetry...........................................................................106 Figure 3.3: Active sites of structurally superposed ABDC and E. coli AAT ..............................107 Figure 3.4: Ribbon representation of the ABDC dodecamer with each monomer labeled. ........108 Figure 3.5: Schematic illustrating the symmetry relationships between monomers in the ABDC particle.........................................................................................................................109 Figure 4.1: PfTrpB2 monomer with secondary structure elements labeled.................................132 Figure 4.2: A structural superposition between the PfTrpB2 and PfTrpB1 identifies substitutions among residues surrounding the PLP cofactor............................................................133 Figure 4.3: The StTrpAB1 complex has an intramolecular tunnel through which indole is diffused from the α-subunit to the β-subunit...............................................................134 Figure 4.4: The StTrpAB1 complex has an intramolecular tunnel through which indole is diffused from the α-subunit to the β-subunit...............................................................135 Figure 4.5: Spectral changes associated with the sequential addition of 5 mM L-L-cys, and 0.1 mM indole, to a solution containing PfTrpB2.............................................................136 Figure 4.6: Initial rate vs [S] profiles for the L-ser/L-cys + indole condensation catalyzed by PfTrpB1, PfTrpAB1, StTrpAB1, and StTrpB1...........................................................137 1 INTRODUCTION AND LITERATURE REVIEW Pyridoxal-5’-phosphate (PLP) is the phosphate ester and catalytically active form of vitamin B6. Enzymes utilizing PLP as a cofactor are among the most catalytically diverse group of proteins, containing members in five of six (1) Enzyme Commission (2) (EC) groups, which equates to approximately 4% of the reactions catalogued in the EC (3). It is believed

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