title: Development of Saccharomyces cerevisiae reductase YOL151W mutants suitable for chiral alcohol synthesis using an NADH cofactor regeneration system.
abstract: The aldo-keto reductases catalyze reduction reactions using various aliphatic and aromatic aldehydes/ketones. Most reductases require NADPH exclusively as their cofactors. However, NADPH is much more expensive and unstable than NADH. In this study, we attempted to change the five amino acid residues that interact with the 2'-phosphate group of the adenosine ribose of NADPH. These residues were selected based on a docking model of the YOL151W reductase and were substituted with other amino acids to develop NADH-utilizing enzymes. Ten mutants were constructed by site-directed mutagenesis and expressed in Escherichia coli. Among them, four mutants showed higher reductase activities than wild-type when using the NADH cofactor. Analysis of the kinetic parameters for the wild type and mutants indicated that the kcat/Km value of the Asn9Glu mutant toward NADH increased 3-fold. A docking model was used to show that the carboxyl group of Glu 9 of the mutant formed an additional hydrogen bond with the 2'-hydroxyl group of adenosine ribose. The Asn9Glu mutant was able to produce (R)-ethyl-4-chloro-3-hydroxyl butanoate rapidly when using the NADH regeneration system.
authors: Division of Biotechnology, The Catholic University of Korea, Bucheon, Korea.
authors: Shin Ah
authors: SA
authors: Yoon
authors: Jihye
authors: J
authors: Jung
authors: Seongsoon
authors: S
authors: Park
authors: Hyung Kwoun
authors: HK
authors: Kim
mesh_terms: D000438:Alcohols / Q000378:metabolism*; D019943:Amino Acid Substitution; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000438:Alcohols; D003067:Coenzymes; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D010088:Oxidoreductases; C432953:GRE2 protein, S cerevisiae