Over-Expression and Characterization of a Glyoxalase 2 Like Enzyme
Total Page：16
File Type：pdf, Size：1020Kb
MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Pattraranee Limphong Candidate for the Degree: Doctor of Philosophy _______________________________________________ Director Dr. Christopher A. Makaroff _______________________________________________ Reader Dr. Michael W. Crowder _______________________________________________ Reader Dr. John W. Hawes _______________________________________________ Reader Dr. Neil D. Danielson _______________________________________________ Graduate School Representative Dr. Qingshun Li ABSTRACT OVER-EXPRESSION AND CHARACTERIZATION OF A GLYOXALASE 2 LIKE ENZYME By Pattraranee Limphong This dissertation consists of seven chapters to better probe and understand the structure and function of a glyoxalase 2 (GLX2) like enzyme from Arabidopsis thaliana and human. Chapter 1 provides a general background of the glyoxalase system followed by the physiological roles of the glyoxalase system, and inhibitors of the glyoxalase enzymes, glyoxalase 1 and glyoxalase 2. Chapter 2 focuses on over-expression and characterization of GLX2-1 (putative glyoxalase 2, isozyme1) from Arabidopsis thaliana. Metal analyses, kinetics, and spectroscopic studies suggested that GLX2-1 contains a dinuclear metal binding site but is not a glyoxalase 2. Chapter 3 investigates the physiological role of GLX2-1. A commercial survey substrate system and β-lactamase substrates were used. The results showed that GLX2-1 exhibits β-lactamase activity. Chapter 4 presents the results of experiments to convert inactive GLX2-1 into an active GLX2 enzyme. Substrate binding residues were mutated at positions 225, 253, 255, 332, and 335 in GLX2-1. The results showed that the R253H GLX2-1 does hydrolyze S-D-lactoylglutathione (SLG) when the substrate binding ligands were generated on the R253H GLX2-1 enzyme. Chapter 5 provides detailed structural information on the human GLX2 metal center and insights concerning the structure and kinetic mechanism of the enzyme. Biochemical and spectroscopic studies showed that human GLX2 contains an Fe(II)Zn(II) center but is active as a mononuclear Zn(II) enzyme. Chapter 6 explains why purified A. thaliana GLX2-2 (glyoxalase 2, isozyme 2) samples contain different metal content and how changes in metal composition affect the structure and activity of the enzyme. Metal analysis and spectroscopic studies provided evidence of different metal centers of each fraction of purified GLX2-2 enzyme. The individual steady state kinetic constants varied among the fractionated species. The data demonstrate that the different reported metal content of purified GLX2-2 samples can be explained by the choice of column fractions that were combined and concentrated. Chapter 7 summarizes the overall conclusions of dissertation. There is also some discussion about additional questions/issues still remaining, and future studies for glyoxalase 2 enzymes. OVER-EXPRESSION AND CHARACTERIZATION OF A GLYOXALASE 2 LIKE ENZYME A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry by Pattraranee Limphong Miami University Oxford, Ohio 2009 Dissertation Directors: Dr. Christopher A. Makaroff and Dr. Michael W. Crowder TABLE OF CONTENTS Chapter 1 Introduction of the Glyoxalase system 1 Chapter 2 Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site 17 but is not a glyoxalase 2 Chapter 3 Arabidopsis thaliana Mitochondrial Glyoxalase 2-1 Exhibits 44 β-Lactamase Activity Chapter 4 Converting GLX2-1 into an active glyoxalase II 60 Chapter 5 Human GLX2 contains an Fe(II)Zn(II) center but is active as 88 a mononuclear Zn(II) enzyme Chapter 6 Metal content of recombinant A. thaliana GLX2-2 is determined 117 by which column fractions are pooled and concentrated Chapter 7 Concluding Remarks 142 ii LIST OF FIGURES Chapter 1 Figure 1: The glyoxalase system. 14 Figure 2: Alignment of predicted plant glyoxalase ІІ’s from A. thaliana. 15 Figure 3: Structure of mitochondrial GLX2-5 and human GLX2. 16 Chapter 2 Figure 1: Structure of mitochondrial GLX2-5. 39 Figure 2: Alignment of predicted plant glyoxalase ІІ’s. 40 Figure 3: Fluorescence emission spectra of wild-type, Fe-added, 41 Zn(II)-added, and FeZn(II)-added GLX2-1. Figure 4: UV-Vis spectrum of 1.2 mM as-isolated GLX2-1 42 in 10 mM MOPS, pH 7.2. Figure 5: EPR spectra of GLX2-1. 43 Chapter 3 Figure 1: Active site structures of human Glx2 and 56 metallo-β-lactamase L1. Figure 2: Nitrocefin hydrolysis by GLX2-1 and by prep minus GLX2-1. 57 Figure 3: Stopped-flow UV-Vis studies with nitrocefin and GLX2-1. 57 Figure 4: Alignment of predicted plant mitochrondria GLX2-1 and 58 GLX2-5 from A. thaliana and GLX2-1 from Brassica. Figure 5: Nucleotide and deduced amino acid sequence of 59 Brassica GLX2-1. iii Chapter 4 Figure 1: Crystal structure of Arabidopsis thaliana GLX2-5 80 and human GLX2. Figure 2: Computational model of the active site of GLX2-1 81 overlapped with the active site of GLX2-5. Figure 3: GLX2-1 model generated using Swiss-Model program. 82 Figure 4: Alignment of predicted plant glyoxalase ІІ’s from A. thaliana. 83 Figure 5: 1H NMR spectrum of the 2Co-R253H mutant of GLX2-1 84 in 10 mM MOPS, pH 7.2, containing 10% D2O. Figure 6: Fluorescence emission spectra of wild-type GLX2-1 85 and GLX2-1 mutants. Figure 7: EPR spectra from H253R GLX2-5. (top) As-isolated 86 H253R GLX2-5. (bottom) As-isolated H253R GLX2-5 + 3eq. Fe and Zn(II). Figure 8: Fluorescence emission spectra of wild-type GLX2-5 87 and the H253R mutant of GLX2-5. Chapter 5 Figure 1: Proposed active site of human GLX2. 110 Figure 2: Stopped-flow kinetic studies of the reaction of human 111 GLX2 analogs with SLG at 2 oC. Figure 3: 1H NMR spectra of human GLX2 analogs in 10 mM MOPS. 112 Figure 4: UV-Vis spectra of GLX2-Comin and GLX2-Comin 113 + 1 equivalent of Zn(II). Figure 5: EPR spectra of human GLX2 analogs. (A) GLX2-LB and 114 (B) GLX2-LB+1.5Zn+1.5 Fe. Figure 6: EPR spectra of human GLX2 containing Co(II). 115 iv Chapter 6 Figure 1: SDS-PAGE of column fractions containing GLX2-2. 135 Figure 2: The metal content column fractions containing recombinant 136 GLX2-2. Figure 3: Kinetic parameters of GLX2-2. 137 Figure 4: UV-Vis spectra of GLX2-2 samples. 138 Figure 5: EPR spectra from GLX2-2. 139 Figure 6: Fe and paramagnet content of GLX2-2 fractions. 140 Figure 7: 1H NMR spectra of GLX2-2 samples. 141 Chapter 7 Figure 1: Alignment of predicted plant glyoxalase ІІ’s from A. thaliana. 147 v LIST OF TABLES Chapter 2 Table 1: Metal content of GLX2-1 analogs. 38 Chapter 4 Table 1: Oligonucleotide primers for site-directed mutagenesis 78 Table 2: Metal content and steady-state kinetic constants for wild type 79 and mutant GLX2-1 and GLX2-5 analogs. Chapter 5 Table 1: Metal content and steady-state kinetic constants 108 for human GLX2 analogs Chapter 6 Table 1: Purification of recombinant Arabidopsis thaliana GLX2-2 133 Table 2: Metal content and steady-state kinetic constants for column 134 fractions containing recombinant GLX2-2. vi Acknowledgements The author would like to thank those who help with this work. Profound gratitude goes to Dr. Chris Makaroff and Dr. Michael Crowder for their guidance, support, assistance, and encouragement throughout this dissertation work. I would like to thank Dr. Brian Bennett at Medical College at Wisconsin, Milwaukee, for analyzing EPR spectra. I would like to acknowledge interesting and helpful discussions with Dr. John Hawes, Dr. Shisong Ni, Dr. Zhenxin Hu, Dr. Meghan Holdorf. Also, I thank Aleks Pisarenko for ICP troubleshooting and Sriram Devanathan for his help with general lab questions. My undergraduate student co-workers, Ross McKinney, Nicole Adams, Chris Traner, and Ankur Patel, are thanked for helping the author with demanding laboratory work. I would like to thank my committee member: Drs. John Hawes, Neil Danielson, and Paul James for their support during my studies in Miami University, and also Dr. Qingshun Li for substituting on my committee for my final defense. I would also like to thank the Department of Chemistry and Biochemistry at Miami University for financial support and CMSB for a research fellowship for the last year in the graduate school. I would like to thank Patrick Hensley and all of the students in Crowder’s and Makaroff’s labs for teaching me experimental techniques and helping me in many ways. Finally, I would like to dedicate this work to my mother, Ms. Poolsri Poomphoung, and to my sisters, Ms. Sopita Limphong and Ms. Pattareeya Limphong. All of my love also goes to my parents, without them, I could never have accomplished so much, and I am forever grateful for them. Also I would like to thank my cat, Lucky, for her company to pass the lonely life in graduate school. vii Chapter 1 Introduction The Glyoxalase System The glyoxalase system is made up of two enzymes that detoxify methylglyoxal and the other reactive oxoaldehydes, which are produced as a normal part of metabolism. This system is comprised of two enzymes: glyoxalase 1 (GLX1; lactoylglutathione lyase, EC 4.4.1.5) and glyoxalase 2 (GLX2; hydroxyacylglutathione hydrolase, EC 3.1.2.6). GLX1 catalyzes the isomerization of the hemithioacetal adduct that is formed by the spontaneous reaction between reduced glutathione (GSH) and a 2-oxoaldehyde into S-2-hydroxyacylglutathione (1, 2). The most prevalent 2-oxoaldehyde is methylglyoxal (MG), and its hemithioacetal is S-D- lactoylglutathione (SLG). GLX2 then hydrolyzes SLG and produces D-lactate and GSH (Figure 1) (1, 3). MG, thought to be the primary physiological substrate of GLX1, is formed in many biological processes including glycolysis, acetone and threonine catabolism, and nonenzymatically from triose phosphates (2, 4, 5).