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Substitution of the critical methionine residues in Trigonopsis variabilisD-amino acid oxidase with leucine enhances its resistance to hydrogen peroxide

Abstract Each of the six oxidative-sensitive methionine residues in Trigonopsis variabilisD-amino acid oxidase (DAAO) was changed to leucine by site-directed mutagenesis. The wild-type and mutant enzymes with an apparent molecular mass of about 39.3 kDa were expressed in Escherichia coli. The specif...

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Bibliographic Details
Published in:FEMS microbiology letters 2000-05, Vol.186 (2), p.215-219
Main Authors: Ju, Sheau-Shya, Lin, Long-Liu, Chien, Hungchien Roger, Hsu, Wen-Hwei
Format: Article
Language:English
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Summary:Abstract Each of the six oxidative-sensitive methionine residues in Trigonopsis variabilisD-amino acid oxidase (DAAO) was changed to leucine by site-directed mutagenesis. The wild-type and mutant enzymes with an apparent molecular mass of about 39.3 kDa were expressed in Escherichia coli. The specific activity of four mutant DAAOs (Met104Leu, Met226Leu, Met245Leu, and Met339Leu) was decreased by more than 96%, while Met156Leu and Met209Leu showed about 23% and 96% higher activity, respectively, than the wild-type enzyme. The kinetic parameters of the two more active enzymes were determined and a 2.2-fold increase in Km was observed for Met209Leu. Comparison of Met156Leu and wild-type DAAO revealed a 95% increase in kcat/Km. Met156Leu, Met209Leu, and Met226Leu were resistant to inactivation by 50 mM H2O2. The other three mutant DAAOs were also slightly more resistant than the wild-type enzyme to chemical oxidation. These observations indicate that the oxidative stability in T. variabilis DAAO can be improved by substitution of methionine residues with leucine.
ISSN:0378-1097
1574-6968
DOI:10.1111/j.1574-6968.2000.tb09107.x