cDNA cloning of novel aldehyde reductase gene from yeast and its application to chiral alcohol synthesis

New aldehyde reductases (AR), ARII and ARIIIwhich reduce ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl 4-chloro-3-hydroxybutanoate (CHBE)，with NADPH as a cofactor，从Sporobolomyces salmonicolor AKU4429. ARII catalyzed the stereospecific reductionof 4-COBE to (S)-CHBE (92.7% enantiomeric excess (e.e.)), in contrast，ARIII reduced 4-COBE to R-CHBE (38.4% e.e.). ARII reduced aliphatic and aromatic aldehydes，and carbonyl compounds such as camphorquinone，但不接受作为一个substrate. The enzyme是一个monomer protein with a relative molecularmass of 34,000. Its isoelectric point is 5.0. The NH2-terminal amino acid sequence of ARII isdifferent from that of ARI，which catalyzes the stereospecific reduction of 4-COBB to R-CHBE (100% e.e.). We cloned andsequenced the gene encoding an NADPH-dependent ARII from S. salmonicolor AKU4429. the ARII genecomprises 1,032 bp, is interrupted by 4 introns，and codes a polypeptide of 37,315 Da. The deduced amino acid…sequence showed significant similarity to that of members of the mammalian3 -hydroxysteroid dehydrogenase/plant dihydroflavonol 4-reductase superfamily，but not to those of the aldo-keto reductase superfamily or to that of ARI previouslyisolated from the same organism. ARII protein was overproduced in Escherichia coli about2000 -fold per g cells，enzyme expressed in E. coli was已确定homogeneity,and showed the same catalytic properties as ARII purified from S. salmonicolor. To examine thecontribution of the dinucleotide-binding motif, G - D219 - D2- x - x -G - 222 - D2- x - x - a - D225 - D2，located in the NH2-terminal region, for ARII catalysis，we exchanged three amino acid residues in the motif，4-COBE was . the substrate inhibition of the enzyme by 4absent in the G - D219 - D2→A and G - 222 - D2→A mutant enzymes. the A - 225 - D2→G mutant enzymecould reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH. Less

New aldehyde reductases (AR), ARII and ARIII, which reduce ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl 4-chloro-3-hydroxybutanoate (CHBE), with NADPH as a cofactor, were purified from Sporobolomyces salmonicolor AKU4429. ARII catalyzed the stereospecific reduction of 4-COBE to (S)-CHBE (92.7% enantiomeric excess (e.e.)), in contrast, ARIII reduced 4-COBE to R-CHBE (38.4% e.e.). ARII reduced aliphatic and aromatic aldehydes, and carbonyl compounds such as camphorquinone, but did not accept aldose as a substrate. The enzyme is a monomer protein with a relative molecular mass of 34,000. Its isoelectric point is 5.0. The NH2-terminal amino acid sequence of ARII is different from that of ARI, which catalyzes the stereospecific reduction of 4-COBB to R-CHBE (100% e.e.). We cloned and sequenced the gene encoding an NADPH-dependent ARII from S. salmonicolor AKU4429. The ARII gene comprises 1,032 bp, is interrupted by 4 introns, and encodes a polypeptide of 37,315 Da. The deduced amino acid … More sequence showed significant similarity to that of members of the mammalian 3β-hydroxysteroid dehydrogenase/plant dihydroflavonol 4-reductase superfamily, but not to those of members of the aldo-keto reductase superfamily or to that of ARI previously isolated from the same organism. The ARII protein was overproduced in Escherichia coli about 2,000-fold per g cells, compared to in the original yeast cells. The enzyme expressed in E. coli was purified to homogeneity, and showed the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif, GィイD219ィエD2-X-X-GィイD222ィエD2-X-X-AィイD225ィエD2, located in the NH2-terminal region, for ARII catalysis, we exchanged three amino acid residues in the motif, and purified the respective mutant enzymes. The substrate inhibition of the enzyme by 4-COBE was absent in the GィイD219ィエD2 → A and GィイD222ィエD2 → A mutant enzymes. The AィイD225ィエD2 → G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH. Less