title: [Broader substrate specifity of Candida parapsilosis SCR II for catalyzing acetophenone derivatives by site-directed mutagenesis].
abstract: OBJECTIVE
  The function for catalyzing acetophenone derivatives of short-chain carbonyl reductase II (SCR II) from Candida parapsilosis was modified by site-directed mutagenesis.
  
  
  METHODS
  An important site (E228) was selected for mutagenesis through amino acid sequence and protein structure alignment, and the corresponding variant E228S was constructed in E. coli. Using the acetophenone derivatives as substrates, we determined specific activities and biotransformation function of the variant.
  
  
  RESULTS
  The specific activity of the variant E228S was reduced to 25% of the wild type for 2-hydroxyacetophenone reduction. However, it increased approximately 7-20 times for acetophenone, 4'-methylacetophenone and 4'-chloroacetophenone. The biotransformation results showed that the variant catalyzed the transformation of (S)-1-phenyl-1,2-ethanediol in a yield of less than 10%, while exhibited excellent performance to afford (R)-1-phenethylethanol, (R )-1-(4-methylpheyl) ethanone and (R)-1-(4-chlorophenyl) ethanol from acetophenone, 4'-methylacetophenone and 4'-chloroacetophenone with high optical purity of 99% in a yield of above 80%.
  
  
  CONCLUSIONS
  We broadened the substrate specificity and catalytic function of SCR II by replacement of the critical amino acid (E228) inside the substrate binding pocket, which provided a novel approach for rational modification of short-chain carbonyl reductases, making it as a potential tool for asymmetric reduction and chiral alcohol preparation.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. zhangbotao1986@yahoo.com.cn
authors.forename: Botao
authors.initials: B
authors.lastname: Zhang
authors.forename: Rongzhen
authors.initials: R
authors.lastname: Zhang
authors.forename: Shanshan
authors.initials: S
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000098:Acetophenones / Q000737:chemistry / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002175:Candida / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000098:Acetophenones; D005656:Fungal Proteins; D000429:Alcohol Oxidoreductases