title: Development of a bioconversion system using Saccharomyces cerevisiae Reductase YOR120W and Bacillus subtilis glucose dehydrogenase for chiral alcohol synthesis.
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.
authors.affiliation: Division of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Republic of Korea.
authors.forename: Shin Ah
authors.initials: SA
authors.lastname: Yoon
authors.forename: Hyung Kwoun
authors.initials: HK
authors.lastname: Kim
mesh_terms: D000090:Acetoacetates / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002087:Butyrates / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D004252:DNA Mutational Analysis; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D000090:Acetoacetates; D002087:Butyrates; D003067:Coenzymes; C121244:ethyl 4-chloro-3-hydroxybutanoate; D009249:NADP; C111982:ethyl 4-chloro-3-oxobutanoate; D010088:Oxidoreductases; D042843:Glucose 1-Dehydrogenase