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 variabilis D-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 (Met 104Leu, Met 226Leu, Met 245Leu, and Met 339Leu) was decreased by more than 96%, while Met 156Leu and Met 209Leu 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 K m was observed for Met 209Leu. Comparison of Met 156Leu and wild-type DAAO revealed a 95% increase in k cat/ K m. Met 156Leu, Met 209Leu, and Met 226Leu were resistant to inactivation by 50 mM H 2O 2. 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.