Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of a Bioconversion System Using Saccharomyces cerevisiae Reductase YOR120W and Bacillus subtilis Glucose Dehydrogenase for Chiral Alcohol Synthesis

  • Yoon, Shin Ah (Division of Biotechnology, The Catholic University of Korea) ;
  • Kim, Hyung Kwoun (Division of Biotechnology, The Catholic University of Korea)
  • Received : 2013.05.14
  • Accepted : 2013.06.13
  • Published : 2013.10.28
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Abstract

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.

Keywords

References

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