title: Substitution of the critical methionine residues in trigonopsis variabilis D-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(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.
authors.affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
authors.forename: S S
authors.initials: SS
authors.lastname: Ju
authors.forename: L L
authors.initials: LL
authors.lastname: Lin
authors.forename: H R
authors.initials: HR
authors.lastname: Chien
authors.forename: W H
authors.initials: WH
authors.lastname: Hsu
mesh_terms: D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D007930:Leucine; D008715:Methionine; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D008715:Methionine; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase; D007930:Leucine