PURIFICATION AND PROPERTIES OF 2 OXIDOREDUCTASES CATALYZING THE ENANTIOSELECTIVE REDUCTION OF DIACETYL AND OTHER DIKETONES FROM BAKERS-YEAST

The NADPH‐linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enationselective reduction of diacetyl (2,3‐butanedione) to (S)‐ and (R)‐acetoin (3‐hydroxy‐2‐butanone) [so‐called (S)‐ and (R)‐diacetyl reductases] (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity. The clean‐up procedures comprising streptomycin sulfate treatment, Sephadex G‐25 filtration, DEAE‐Sepharose CL‐6B columm chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120‐fold and 368‐fold purifications, respectively. The relative molecular mass of the (R)‐diacetyl reductase, estimated by means of HPLC filtration on Zorbax GF 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36000, The (R)‐enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5‐, C6‐2,3‐diketones, 1,2‐cyclohexanedione, 2‐oxo aldehydes and short‐chain 2‐ and 3‐oxo esters as substrates. The enzyme was characterized by high enantioselectivity and regiospecificity. The Km values for diacetyl and 2,3‐pentanedione were determined as 2.0 mM. The Mr of the (S)‐diacetyl reductase was determined as 75000 by means of HPLC filtration on Zorbax GF 250. The enzyme decomposed into subunits of Mr 48000 and 24000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH was 6.9. the purified (S)‐enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3‐, 2,4‐ and 2.5‐diketones, 2‐oxo aldehydes, 1,2‐cyclohexanedione and methyl ketones as well as 3‐, 4‐ and 5‐oxo esters. The 2.3‐ and 2,4‐diketones are transformed to the corresponding (S)‐2‐hydroxy ketones; 2,5‐hexanedione, however, was reduced to (S,S)‐2,5‐hexanediol. The Km values for diacetyl and 2,3‐pentanedione were estimated as 2.3 and 1.5 mM, respectively. Further characterization of the (S)‐diacetyl reductase revealed that it is identical with the so‐called ‘(S)‐enzyme’, involved in the enantioselective reduction of 3‐, 4‐ and 5‐oxo esters in baker's yeast. Copyright © 1990, Wiley Blackwell. All rights reserved