Open AccessSubstitution of the critical methionine residues in Trigonopsis variabilis D ‐amino acid oxidase with leucine enhances its resistance to hydrogen peroxide
Author(s) -
Ju SheauShya,
Lin LongLiu,
Chien Hungchien Roger,
Hsu WenHwei
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb09107.x
Subject(s) - d amino acid oxidase , methionine , leucine , biochemistry , chemistry , enzyme , site directed mutagenesis , amino acid , enzyme kinetics , oxidase test , hydrogen peroxide , wild type , mutant , mutagenesis , active site , gene
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 104 Leu, Met 226 Leu, Met 245 Leu, and Met 339 Leu) was decreased by more than 96%, while Met 156 Leu and Met 209 Leu 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 209 Leu. Comparison of Met 156 Leu and wild‐type DAAO revealed a 95% increase in k cat / K m . Met 156 Leu, Met 209 Leu, and Met 226 Leu were resistant to inactivation by 50 mM H 2 O 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.