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KMID : 0545120130230101395
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Journal of Microbiology and Biotechnology
2013 Volume.23 No. 10 p.1395 ~ p.1402
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Development of a Bioconversion System Using Saccharomyces cerevisiae Reductase YOR120W and Bacillus subtilis Glucose Dehydrogenase for Chiral Alcohol Synthesis
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Yoon Shin-Ah
Kim Hyung-Kwoun
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Abstract
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Reductases convert some achiral ketone compounds into chiral alcohols, which are important materials for the synthesis of chiral drugs. The Saccharomyces cerevisiae reductase YOR120W converts ethyl-4-chloro-3-oxobutanoate (ECOB) enantioselectively into (R)-ethyl-4-chloro-3- hydroxybutanoate ((R)-ECHB), an intermediate of a pharmaceutical. As YOR120W requires NADPH as a cofactor for the reduction reaction, a cofactor recycling system using Bacillus subtilis glucose dehydrogenase was employed. Using this coupling reaction system, 100 mM ECOB was converted to (R)-ECHB. A homology modeling and site-directed mutagenesis experiment were performed to determine the NADPH-binding site of YOR120W. Four residues (Q29, K264, N267, and R270) were suggested by homology and docking modeling to interact directly with 2’-phosphate of NADPH. Among them, two positively charged residues (K264 and R270) were experimentally demonstrated to be necessary for NADPH 2’-phosphate binding. A mutant enzyme (Q29E) showed an enhanced enantiomeric excess value compared with that of the wild-type enzyme.
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KEYWORD
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Reductase, Chiral compound, Coupling reaction, NADPH regeneration
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