Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3‐oxo, 4‐oxo and 5‐oxo esters in baker's yeast

Abstract

Two NADPH‐dependent oxidoreductases catalyzing the enantioselective reduction of 3‐oxo esters to (S)‐ and (R)‐3‐hydroxy acid esters, [hereafter called (S)‐ and (R)‐enzymes] have been purified 121‐ and 332‐fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G‐25 filtration, DEAE‐Sepharose CL‐6B chromatography, Sephadex G‐150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)‐enzyme was estimated to be 48000–50000 on Sephadex G‐150 column chromatography and 48000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3‐oxo esters, 4‐oxo and 5‐oxo acids and esters enantioselectively to (S)‐hydroxy compounds in the presence of NADPH. The Km, values for ethyl 3‐oxobutyrate, ethyl 3‐oxohexanoate, 4‐oxopentanoic and 5‐oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)‐enzyme, estimated by means of column chromatography on Sepharose 6B, was 800 000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200000 and 210000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3‐oxo esters to (R)‐hydroxy esters, using NADPH for coenzyme. Km, values for ethyl 3‐oxobutyrate and ethyl 3‐oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)‐enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr, 2.4 × 106) showed no activity in catalyzing the reduction of 3‐oxo esters. Copyright © 1988, Wiley Blackwell. All rights reserved