Abstract
Diethyl 2-methyl-3-oxosuccinate reductase was purified from cells of Saccharomyces fermentati by streptomycin treatment, Sephadex G-50 gel filtration, a first DEAE-cellulose chromatography, a first Toyopearl HW-60F gel filtration, a second DEAE-cellulose chromatography and a second Toyopearl HW-60F gel filtration. The enzyme was purified 169-fold and gave one band in sodium dodecyl sulfate(SDS)/polyacrylamide-gel electrophoresis. The molecular weight of the purified enzyme was 61, 000 to 63, 000. The enzyme was NADPH-dependent and had maximum activity at pH 6.0 and 50°C. It was stable at pH 7 to 8. The Km values at pH 7.0 were 1.25mM for diethyl 2-methyl-3-oxosuccinate and 111 mM for the α-methyl β-hydroxy esters syn-(2) and anti-(3). The enzyme reduced methyl 2-methyl-3-oxosuccinate, but did not act on other synthetic substrates.
The optical yields of the products syn-(2) and anti-(3) reduced by the purified enzyme were more than 99% enantiomer excess (e.e.) by measurement of the corresponding (+)-MTPA esters[syn-(10) and anti-(11)] on a 400 MHz NMR instrument.
