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

Each of the six oxidative-sensitive methionine residues in Trigonopsis vari abilis D-amino acid oxidase (DAAO) was changed to leucine by site-directed mutagenesis. The wild-type and mutant enzymes with an apparent molecular ma ss of about 39.3 kDa were expressed in Escherichia coli. The specific activ ity of four mutant DAAOs (Met(104)Leu, Met(226)Leu, Met(245)Leu, and Met(33 9)Leu) was decreased by more than 96% while Met(156)Leu and Met(209)Leu sho wed about 23% and 96% higher activity, respectively, than the wild-type enz yme. The kinetic parameters of the two mure active enzymes were determined and a 2.2-fold increase in K-m was observed for Met(209)Leu. Comparison of Met(156)Leu and wild-type DAAO revealed a 95% increase in k(cat)/K-m. Met(1 56)Leu, Met(209)Leu, and Met(226)Leu were resistant to inactivation by 50 m M H2O2 The other three mutant DAAOs were also slightly more resistant than the wild-type enzyme to chemical oxidation. These observations indicate tha t the oxidative stability in T. variabilis DAAO can be improved by substitu tion of methionine residues with leucine. (C) 2000 Published by Elsevier Sc ience B.V. All rights reserved.