Cloning, sequence analysis, and expression in Escherichia coli of the geneencoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp strain ST-10

The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from the genomic DNA Of the styre ne-assimilating Corynebacterium sp. strain ST-10. The gene contained an ope ning reading frame consisting of 1,158 nucleotides corresponding to 385 ami no acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrop horesis, The enzyme was sufficiently expressed in recombinant Escherichia c oli cells for practical use and purified to homogeneity by three-column chr omatography steps. The predicted amino acid sequence displayed only 20-29% identity with zinc-containing, NAD(+)-dependent, long-chain alcohol dehydro genases. Nevertheless, the probable NAD(+)- and zinc-binding sites are cons erved although one of the three catalytic zinc-binding residues of the zinc -containing, long-chain alcohol dehydrogenases was substituted by Asp in PA R. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme wa s considered as a new member of zinc-containing, long-chain alcohol dehydro genases with a particular and broad substrate specificity.