Purification and Characterization of Ethyl 2-Methyl-3-oxobutanoate Reductase from Klebsiella pneumoniae IF03319.
An enzyme that catalyzes a reduction of ethyl 2-methyl-3-oxobutanoate (1) to ethyl (2R,3S) 3-hydroxy- 2-methylbutanoate was found in Klebsiella pneumoniae IFO 3319 cells. The enzyme was isolated from the cells and purified 250-fold by ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and gel filtration. The purified enzyme was found to be a monomer protein with a molecular weight of approximately 31,000 and an isoelectric point of 6.2. It was NADPH-dependent and had maximum activity at pH 7.0 and 45°C for the reduction and at pH 10.0 and 45°C for oxidation. The Km's at pH 7.0 were 5.6 mM for 1 and 12.5 mM for benzyl 2-methyl-3-oxobutanoate, respectively. Esters of 2-oxocycloalkane carboxylic acids as well as esters of 2-methyl-3-oxobutanoic acid served as substrates, and the corresponding reduced products were obtained with high stereoselectivity.
a Integrated Technology Laboratories , Takeda Chemical Industries Ltd. , Wadai 10, Tsukuba, Ibaraki 300-42 , Japan.
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Miya
a Integrated Technology Laboratories , Takeda Chemical Industries Ltd. , Wadai 10, Tsukuba, Ibaraki 300-42 , Japan.
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Kawada
a Integrated Technology Laboratories , Takeda Chemical Industries Ltd. , Wadai 10, Tsukuba, Ibaraki 300-42 , Japan.
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Sugiyama
Klebsiella pneumoniae IFO 3319; ethyl (2R,3S) 3-hydroxy-2-methylbutanoate; ethyl 2-methyl-3-oxobutanoate reductase; stereoselective reduction