Purification and characterization of two alpha-keto ester reductases from Streptomyces thermocyaneoviolaceus IFO 14271.
Two NADPH-dependent alpha-keto ester reductases (Streptomyces thermocyaneoviolaceus keto ester reductase, STKER-II and -III) were purified from S. thermocyaneoviolaceus IFO 14271, one of thermophilic actinomycetes. The molecular masses of native STKER-II and -III were estimated to be 60 kDa and 70 kDa by gel filtration chromatography, respectively. These enzymes were both homodimers, with 29-kDa and 30-kDa subunit molecular masses based on SDS polyacrylamide gel electrophoresis. STKER-II and -III were stable from pH 7.0 to 10.0 and pH 5.5 to 9.0, respectively. Ethyl 3-methyl-2-oxobutanoate was reduced by both enzymes isolated to the corresponding (R)-hydroxy ester with excellent enantiomeric excess. STKER-III showed high stereoselectivity for the reduction of bulky substrates, while the selectivity of the STKER-II-catalyzed reduction was low except for ethyl 3-methyl-2-hydrox-ybutanoate. Both enzymes had small Km values toward aliphatic keto esters having a long alkyl chain.
Yamaguchi
Hitomi
H
Institute for Chemical Research, Kyoto University, Uji, Japan.
Nakajima
Nobuyoshi
N
Ishihara
Kohji
K
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008968:Molecular Conformation; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013302:Streptomyces / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003470:Culture Media; D000429:Alcohol Oxidoreductases