Enzymes producing two chiral centers. Part I. Purification and properties of an asymmetric reduction enzyme of 2-methyl-3-oxobutyrate in baker's yeast.

The benzyl 2-methyl-3-hydroxybutyrate dehydrogenase was purified from the cells of baker's yeast by streptomycin treatment, Sephadex G-50 gel filtration, SP-Sephadex C-50 chromatography, and Toyopearl HW-60F gel filtration. The purified enzyme preparation was homogeneous and the molecular weight was about 31, 000 to 32, 000. The enzyme was NADPH-dependent and its maximum activity was at pH 7.0 and 45°C. It was stable between pH 6 and 9. The Km values at pH 7.0 were 0.42mM for benzyl 2-methyl-3-oxobutyrate (1) and 4.2mM for α-methyl β-hydroxy ester [syn-(2) and anti-(3)]. This enzyme reduced only benzyl 2-methyl-3-oxobutyrate (1) but had no effect on other synthetic substrates. The reduced products [syn-(2) and anti(3)] produced by the purified enzyme were identified by 400MHz NMR.