Purification and characterization of NADPH-dependent carbonyl reductase, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, from Candida magnoliae.
A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue Sepharose affinity chromatography. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a 100% enantiomeric excess, which is a useful chiral building block for the chemical synthesis of pharmaceuticals. The relative molecular mass of the enzyme was estimated to be 76,000 on high performance gel filtration chromatography and 32,000 on SDS polyacrylamide gel electrophoresis. The enzyme reduced alpha-, beta-keto esters and conjugated diketones in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by quercetin and HgCl2, but not by EDTA. The N-terminal amino acid sequence of the enzyme showed no apparent similarity with those of other oxidoreductases.
Wada
M
M
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Kataoka
M
M
Kawabata
H
H
Yasohara
Y
Y
Kizaki
N
N
Hasegawa
J
J
Shimizu
S
S
D000090:Acetoacetates / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D002474:Cell-Free System; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D013379:Substrate Specificity
D000090:Acetoacetates; D004791:Enzyme Inhibitors; C111982:ethyl 4-chloro-3-oxobutanoate; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase