Site-directed Mutagenesis of Two Zinc-binding Centers of the NADH-dependent Phenylacetaldehyde Reductase from Styrene-assimilating Corynebacterium sp. Strain ST-10

• Published in: Bioscience, biotechnology, and biochemistry Vol. 63; no. 12; pp. 2216 - 2218
• Main Authors: WANG, Jiu-Cun, SAKAKIBARA, Mikio, MATSUDA, Michiko, ITOH, Nobuya
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.01.1999; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biotechnology; Corynebacterium - enzymology; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; long-chain alcohol dehydrogenase; Methods. Procedures. Technologies; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidoreductases; phenylacetaldehyde reductase; Protein engineering; site-directed mutagenesis; Styrene - metabolism; Zinc - metabolism; zinc-binding site; zinc-containing; Enzyme; Site directed mutagenesis; Binding site; Zinc; Corynebacterium; Enzymatic activity; Structure activity relation; Substrate specificity; Bacteria; Actinomycetes; Corynebacteriaceae; Oxidoreductases; Mutation; Aminoacid sequence
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.63.2216

Phenylacetaldehyde reductase (PAR) with a unique and wide substrate range from styrene-assimilating Corynebacterium sp. strain ST-10, which is a useful biocatalyst producing chiral alcohols, has been found to belong to a family of zinc-containing, long-chain alcohol dehydrogenases (ADHs) on the basis of the primary structure similarity. The enzyme contains 2 moles of zinc per mole of subunit. The amino acid residues assumed to be three catalytic and four structural zinc-binding ligands were characterized by site-directed mutagenesis, compared with other zinc-containing, long-chain ADHs. Sixteen PAR mutants gave measurable but rather low activities toward phenylacetaldehyde, n-hexyl aldehyde, and 2-heptanone, although they maintained the activities of 8 to 16% of that of wild-type PAR for an acetophenone substrate except that the D153N mutant showed quite low activity. The results suggested that the seven residues present in PAR were probably zinc-binding ligands, and mutation in these residues caused a change in activities for some substrates.