Cloning, overexpression, and mutagenesis of the Sporobolomyces salmonicolor AKU4429 gene encoding a new aldehyde reductase, which catalyzes the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate

We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reduc tase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-c hloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate, The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significan t levels of similarity to the amino acid sequences of members of the mammal ian 3 beta-hydroxysteroid dehydrogenase-plant dihydroflavonol 4-reductase s uperfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductas e previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Has himoto, H. Yanase, N, Kato, M. C.-M. Chung, M, Kataoka, and S. Shimizu, App l. Environ. Microbiol. 62:2303-2310, 1996). The ARII protein was overproduc ed in Escherichia coli about 2,000-fold compared to the production in the o riginal yeast cells. The enzyme expressed in E. coli was purified to homoge neity and had the same catalytic properties as ARII purified from S. salmon icolor, To examine the contribution of the dinucleotide-binding motif G(19) -X-X-G(22)-X-X-A(25), which is located in the N-terminal region, during ARI I catalysis, rye replaced three amino acid residues in the motif and purifi ed the resulting mutant enzymes. Substrate inhibition of the G(19)-->A and G(22)-->A mutant enzymes by 4-COBE did not occur, The A(25)-->G mutant enzy me could reduce 4-COBE when NADPH was replaced by an equimolar concentratio n of NADH.