Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding 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 styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrophoresis. The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence displayed only 20-29% identity with zinc-containing, NAD(+)-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD(+)- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate specificity.
Wang
J C
JC
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Fukui University, Japan.
Sakakibara
M
M
Liu
J Q
JQ
Dairi
T
T
Itoh
N
N
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000438:Alcohols / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013343:Styrenes / Q000378:metabolism*; D015032:Zinc / Q000032:analysis
D000438:Alcohols; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D013343:Styrenes; D000429:Alcohol Oxidoreductases; C110191:phenylacetaldehyde reductase; D015032:Zinc