Influence of Arginine 355 and Glutamate 758 on the Substrate Specificities and Catalytic Properties of C. acidovorans Xanthine Dehydrogenase Öffentlichkeit

Li, Yuting (2007)

Permanent URL: https://etd.library.emory.edu/concern/etds/tb09j589z?locale=de
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Abstract

Xanthine dehydrogenase (XDH) was engineered to an aldehyde-oxidizing enzyme (ADH) by a single amino acid mutation with the benefit of identifying the functional roles of active site amino acids in the related XDH and aldehyde oxidase (AO) enzymes that distinguish their respective catalytic activities. Arg355Met C. acidovorans XDH was expressed and purified using pUCP-Nde/P. aeruginosa expression system. Arg355Met XDH exhibits a similar UV-Vis spectrum and CD spectrum with those of the wild-type XDH. On comparison with wild-type enzyme, the mutant enzyme shows 0.4% of the kcat with xanthine, and 3% with hypoxanthine. Interestingly, Arg355Met XDH shows catalytic activity with phenanthridine, which is a substrate of AO but not a substrate of XDH. These results suggest that the Arg355Met XDH has a similar active site structure to that of the wild-type XDH and that Arg355 plays an important role in catalysis of xanthine hydroxylation. The Arg355Met mutation results in the functional conversion of XDH to that of an aldehyde dehydrogenase. Glu758Gln XDH mutant expressed from P. aeruginosa was purified. An imidazole gradient was used for elution from Ni-NTA column during purification to separate the endogenous XDH in P. aeruginosa from the expressed mutant enzyme, because the presence of endogenous XDH will interfere with the kinetic behavior of the expressed enzyme that exhibits a very low catalytic activity. The recombinant mutant enzyme exhibits UV-Vis and CD spectral properties very similar to the wild-type XDH, and kinetic characterization of the Glu758Gln XDH at pH 7.8 reveals only 0.002% of turnover number of the wild-type XDH. The result shows the importance of Glu758 in the catalytic mechanism of xanthine hydroxylation. Combined with previous results from our lab, a catalytic mechanism was proposed with Glu758 as a general base to initiate the reaction by deprotonating the Mo-OH group.

Table of Contents

Chapter I General Introduction 1.1 Molybdenum Hydroxylases 1.2 Xanthine Oxidoreductases 1.2.1 Eukaryotic XOR and Prokaryotic XOR 1.2.2 XOR cofactors 1.2.2.1 Molybdenum Pyranopterin 1.2.2.2 Iron-sulfur centers 1.2.2.3 Flavin Adenine Dinucleotide (FAD) 1.2.3 Properties of XOR 1.2.3.1 UV-Vis and CD Spectral Properties 1.2.3.2 EPR spectroscopy 1.2.4 Kinetics Studies of XOR 1.2.4.1 Steady State Kinetics 1.2.4.2 Reductive Half-reaction 1.2.4.3 Oxidative Half-reaction 1.2.4.4 Electron Transfer in XOR 1.2.4.5 Proposed Catalytic Mechanism 1.2.5 Expression Systems 1.3 Aldehyde Oxidase 1.3.1 Reaction Mechanism of Aldehyde Oxidase 1.4 Dissertation Objectives Chapter 2 Engineering of the C. acidovorans Xanthine Dehydrgenase to a Molybdo-enzyme with Aldehyde Dehydrogenase Functional Properties 2.1 Introduction 2.2 Materials and Methods 2.2.1 Materials 2.2.2 Construction of Vector pMT801 for Arg355Met Expression 2.2.3 Expression and purification of recombinant Arg355Met XDH 2.2.4 Characterization of recombinant Arg355Met XDH 2.2.5 Determination of the product obtained by oxidation of phenanthridine by Arg355Met XDH 2.2.6 Determination of inhibition constant of phenanthridine for WT XDH 2.2.7 Kinetics analysis for reactions of NADH and 6-phenanthridone formation 2.3 Results 2.3.1 Expression and purification of recombinant Arg355Met in P. aeruginosa PAOl-LAC 2.3.2 Characterization of recombinant Arg355Met from P. aeruginosa PAO1- LAC 2.3.2.1 Chemical properties and Spectral properties of recombinant Arg355Met XDH 2.3.2.2 Kinetic parameters of recombinant Arg355Met XDH at pH 7.8 2.3.3 Determination of the product obtained by oxidation of phenanthridine through Arg355Met C. acidovorans XDH 2.3.4 Determination of Inhibition constant of phenanthridine for WT XDH 2.3.5 Kinetics analysis for reactions of NADH and 6-phenanthridone formation 2.4 Discussion 2.5 Conclusion and future work Chapter 3 Investigation of the Catalytic Mechanism of C. acidovorans Xanthine Dehydrogenase: Characterization and Kinetics Studies on the Glu758Gln Active-Site Mutant 3.1 Introduction 3.2 Materials and Methods 3.2.1 Materials 3.2.2 Expression and purification of recombinant Glu758Gln XDH 3.2.3 Characterization of recombinant Glu758Gln XDH 3.3 Results 3.3.1 Expression and purification of recombinant Glu758Gln in P. aeruginosa PAO1-LAC 3.3.2 Characterization of recombinant Glu758Gln from P. aeruginosa PAO1-LAC 3.3.2.1 Spectral properties of recombinant Glu758Gln XDH 3.3.2.2 Chemical properties and kinetic parameters of recombinant Glu758Gln at pH 7.8 3.4 Discussion Chapter 4 General Summary References

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