Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/68152
標題: Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl D-amino acid amidohydrolase by engineering solvent-accessible methionine residues
作者: Chien, H.C.R.
Hsu, C.L.
Hu, H.Y.
Wang, W.C.
Hsu, W.H.
關鍵字: oxidative resistance
N-carbamoyl D-amino acid amidohydrolase
solvent
accessible
site-directed mutagenesis
methionine oxidation
Agrobacterium
site-directed mutagenesis
microbial transformation
conserved
methionines
proteins
thermostability
substitutions
hydantoins
stability
increase
subunit
期刊/報告no:: Biochemical and Biophysical Research Communications, Volume 297, Issue 2, Page(s) 282-287.
摘要: 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 H2O2, 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. (C) 2002 Elsevier Science (USA). All rights reserved.
URI: http://hdl.handle.net/11455/68152
ISSN: 0006-291X
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