Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl D-amino acid amidohydrolase by engineering solvent-accessible methionine residues.
N-Carbamoyl D-amino acid amidohydrolase (D-NCAase) that catalyzes the stereospecific hydrolysis of N-carbamoyl D-amino acids to their corresponding D-amino acids is valuable in pharmaceutical industry. Agrobacterium radiobacter D-NCAase is sensitive to oxidative damage by hydrogen peroxide. To investigate the role of methionine residues in oxidative inactivation, each of the nine methionine residues in A. radiobacter D-NCAase was substituted with leucine, respectively, by site-directed mutagenesis. Except for two mutants (Met5Leu and Met31Leu) with similar activities, seven mutants (Met73Leu, Met167Leu/Met169Leu, Met184Leu, Met220Leu, Met239Leu, Met244Leu, and Met239Leu/Met244Leu) were found to have reduced activities. In the presence of H(2)O(2), three mutants (Met239Leu, Met244Leu, and Met239Leu/Met244Leu) with substitution of highly solvent-accessible methionines by leucines retained their activities. The other mutants were also considerably resistant to chemical oxidation than was the wild-type enzyme. Thus, substitution of solvent-accessible methionine residues with leucine to enhance oxidative stability of D-NCAase is practical but might be with compromised activity.
Roger Chien
Hung-Chien
HC
Department of Microbiology, Chung Shan Medical University, Taichung 402, Taiwan.
Hsu
Chuan-Long
CL
Hu
Hui-Yu
HY
Wang
Wen-Ching
WC
Hsu
Wen-Hwei
WH
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008715:Methionine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D012997:Solvents
D001426:Bacterial Proteins; D016877:Oxidants; D012997:Solvents; D008715:Methionine; D006861:Hydrogen Peroxide; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase