Purification and Characterization of Ethyl 2-Methyl-3-oxobutanoate Reductase from Klebsiella pheumoniae IFO3319

Abstract

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 K_m'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-oxocyclo-alkane 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.