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Over-Expression and Characterization of a Metallohydrolase from Arabidopsis Thaliana

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Over-Expression and Characterization of a Metallohydrolase from Arabidopsis Thaliana ABSTRACT GLYOXALASE 2-2: OVER-EXPRESSION AND CHARACTERIZATION OF A METALLOHYDROLASE FROM ARABIDOPSIS THALIANA By Nathan F. Wenzel In order to investigate the metal binding properties of A. thaliana glyoxalase 2-2, recombinant GLX 2-2 was over-expressed in rich or minimal media containing various amounts of added Zn(II), Fe(II), or Mn(II). The resulting enzymes were purified, characterized, and compared. Kinetic studies and metal analyses of the samples indicated that no significant catalytic differences existed between the enzymes, in spite of the enzymes binding different types and amounts of metal ions. EPR and EXAFS spectroscopies strongly indicated the presence of positive cooperativity of metal binding and of numerous possible dinuclear metal centers, including Fe(III)Zn(II), Fe(III)Fe(II), and Mn(II)Mn(II) centers. The ability to bind various amounts of manganese, iron, and zinc with no apparent change in catalytic activity and the possibility of positive cooperativity in metal binding make A. thaliana glyoxalase 2-2 unique to the family of proteins containing the β-lactamase fold. Glyoxalase 2-2: Over-expression and Characterization of a Metallohydrolase from Arabidopsis thaliana A Thesis Submitted to the Faculty of Miami University In partial fulfillment of The requirement for the degree of Master of Science Department of Chemistry and Biochemistry By Nathan F. Wenzel Miami University Oxford, OH 2003 Advisor________________________ Michael W. Crowder Reader_________________________ Christopher A. Makaroff Reader_________________________ John W. Hawes Reader_________________________ Robert E. Minto Table of Contents 1. Chapter 1 1.1. Introduction: the Glyoxalase system 1 1.2. Physiological Roles of the Glyoxalase system 3 1.3. Inhibitors of the Glyoxalase Enzymes 3 1.4. Glyoxalase 1 5 1.5. Glyoxalase 2 6 1.6. Summary 10 1.7. References 12 2. Chapter 2 2.1. Introduction 17 2.2. Materials and Methods 19 ii 2.2.1. Materials 19 2.2.2. Methods for rich media preparations 19 2.2.2.1. Over-expression and purification 19 2.2.2.2. PIXE sample preparation 20 2.2.2.3. Steady-state kinetics 20 2.2.2.4. Metal analysis 21 2.2.2.5. EPR spectroscopy 21 2.2.2.6. EXAFS sample preparation 21 2.2.3. Methods for minimal media preparation 22 2.2.3.1. Dehua Pei minimal media preparations 22 2.2.3.2. Hepes minimal media preparations 22 2.2.3.3. Steady-state kinetics and metal analysis 23 2.2.3.4. Circular dichroism (CD) and fluorescence spectroscopy 24 iii 2.2.3.5. EPR spectroscopy 24 2.2.3.6. EXAFS spectroscopy 24 2.3. Results and Discussion 25 2.3.1. Rich media GLX 2-2 25 2.3.1.1. Over-expression, purification, and characterization of nma and metal-added GLX 2-2 samples 25 2.3.1.1.1. Over-expression and purification 25 2.3.1.1.2. Determining molar absorptivity (ε280 nm) 25 2.3.1.1.3. Steady-State kinetics 26 2.3.1.1.4. Metal analyses 31 2.3.1.1.5. Electron paramagnetic resonance spectroscopy 40 2.3.1.1.6. EXAFS spectroscopy 48 2.3.2. Minimal media GLX 2-2 55 iv 2.3.2.1. Over-expression, purification, and characterization of nma and metal- added GLX 2-2 samples from minimal media 55 2.3.2.1.1. Over-expression and purification 55 2.3.2.1.2. Steady-state kinetics 56 2.3.2.1.3. Metal analyses 62 2.3.2.1.4. Electron paramagnetic resonance spectroscopy 68 2.3.2.1.5. EXAFS spectroscopy 75 2.3.2.1.6. Circular dichroism 82 2.3.2.1.7. Fluorescence spectroscopy 84 2.4. Conclusions 86 2.5. References 89 v List of Tables Chapter 2 Table 1. Steady-state kinetic data and metal content for rich media GLX 2-2 samples 29 Table 2. Steady-state kinetic data and metal content for rich media GLX 2-2 samples 30 Table 3. Steady-state kinetic data and metal content for rich media GLX 2-2 samples 32 Table 4. Steady-state kinetic data and metal content for rich media GLX 2-2 samples 34 Table 5. Coordination spheres for iron, manganese, and zinc as derived by EXAFS analysis 52 Table 6. Steady-state kinetic data and metal content for Dehua Pei minimal media GLX 2-2 samples 57 Table 7. Steady-state kinetic data and metal content for metal competition GLX 2-2 samples 59 vi Table 8. Steady-state kinetic data and metal content for Hepes minimal media GLX 2-2 samples 61 Table 9. Metal content for high metal bioavailability Hepes minimal media GLX 2-2 samples 67 Table 10. EXAFS model fits 80 vii List of Figures Chapter 1 Figure 1. The Glyoxalase System catalyzes the isomerization of the hemithioacetal intermediate into SLG. 2 Figure 2. Aligned Amino Acid Sequences of GLX 2 9 Chapter 2 Figure 1. SDS-PAGE of a typical column elution profile for recombinant A. thaliana GLX 2-2 (from rich media) 27 Figure 2. Plot of kcat/KM vs. Iron equivalents 35 Figure 3. Plot of kcat/KM vs. Zinc equivalents 37 Figure 4. Plot of kcat/KM vs. Manganese equivalents 38 viii Figure 5. X-Band EPR spectra of three nma GLX 2-2 samples at 4.7 K 41 Figure 6. X-Band EPR spectra of nma GLX 2-2 samples samples at 4.7 K with: a. 1 equivalent H2O2, b. 1 equivalent dithionite, c. nma GLX 2-2 42 Figure 7. X-Band EPR spectra of three Fe-added GLX 2-2 samples at 4.7 K 43 Figure 8. X-Band EPR spectra of three Mn-added GLX 2-2 samples at 4.7 K 44 Figure 9. X-Band EPR spectra of three Zn-added GLX 2-2 samples at 4.7 K 45 Figure 10. Normalized iron, manganese and zinc absorption edges 50 Figure 11. EXAFS spectra and Fourier transform from GLX 2–2 preparations with different ratios of iron, manganese and zinc 53 Figure 12. Model fits of the EXAFS spectra 54 Figure 13. SDS-PAGE gels of Fe-added GLX 2-2 purification 63 ix Figure 14. UV-VIS spectrum of 54.2 µM purple Fe-added GLX 2-2, from Hepes minimal media 66 Figure 15. X-Band EPR spectra of minimal media GLX 2-2 samples at 4.7 K 69 Figure 16. X-Band EPR spectra of Mn-added GLX 2-2 from Hepes minimal media at 4.7 K 71 Figure 17. X-Band EPR spectra of Fe-added GLX 2-2 from Hepes minimal media at 12 K 72 Figure 18. X-Band EPR spectra of Zn-added GLX 2-2 from Hepes minimal media at 4.7 K 73 Figure 19. X-Band EPR spectra of metal competition GLX 2-2 at 4.7 K 74 Figure 20. Normalized iron- and zinc-K-absorption edges of GLX 2-2 77 Figure 21. Iron- and zinc-K-edge EXAFS of GLX 2-2 78 Figure 22. Model fits of GLX 2-2 EXAFS spectra 81 x Figure 23. CD spectra of Hepes minimal media GLX 2-2 samples 83 Figure 24. Fluorescence spectra of Hepes minimal media GLX 2-2 samples 84 Figure 25. Rasmol plot of ligand-metal distance in A. thaliana 87 GLX 2-2. xi Acknowledgements I would like to thank Patrick Crawford and Daniel Sobieski for teaching me many of the techniques used in this thesis. I would also like to thank Anne Carenbauer and Mary Pam Pfister for performing steady-state kinetic studies on the Dehua Pei minimal media GLX 2-2 samples. Many thanks go to Dr. Wolfram Meyer-Klaucke and Oliver Schilling, at the EMBL outstation in Hamburg, Germany, for conducting the EXAFS and PIXE experiments in this thesis. Also, thank you to Melissa Naylor for her supporting steady-state kinetic work. I would like to thank Dr. Makaroff and Dr. Crowder for their invaluable assistance in experimental development, data analysis, and writing. Thank you to Dr. Crowder and the rest of the Crowder lab for providing such a rewarding environment for me to research in and to enjoy my time as a graduate student at Miami. Go team. Finally, I would like to thank my friends and family for their support and help in getting me to blow off some steam every once in a while. Thank you all. xii Chapter 1 1.1 Introduction: The Glyoxalase System The glyoxalase system consists of two enzymes that convert cytotoxic 2-oxoaldehydes into 2-hydroxy acids in the presence of reduced glutathione (Figure 1) (1). These two enzymes are lactoylglutathione lyase (GLX 1, 4.4.1.5) and hydroxyacylglutathione hydrolase (GLX 2, 3.1.2.6). GLX 1 is a Zn(II) containing (or Ni (II) (2)) metalloenzyme that catalyzes the formation of S-D-lactoylglutathione (SLG) from the thiohemiacetal intermediate, which is spontaneously and nonenzymatically formed by methylglyoxal (MG) and reduced glutathione (3). MG, thought to be the primary physiological substrate of GLX 1, is formed in many biological processes including glycolysis, acetone and threonine catabolism, and nonenzymatically from triose phosphates (3-6). MG is both cytotoxic and mutagenic because of its ability to reversibly, or in some cases irreversibly, modify arginine, lysine, and cysteine residues in proteins (7), form modified guanylate residues in DNA and RNA (4), and form inter-strand DNA crosslinks (8). GLX 2 catalyzes the hydrolysis of SLG to form D-lactic acid and regenerate reduced glutathione (3). SLG is cytotoxic because of its ability to inhibit DNA synthesis (4, 9).
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