Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast.
The NADPH-linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enantioselective 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 column 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 36,000. 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 75,000 by means of HPLC filtration of Zorbax GF 250. The enzyme decomposed into subunits of Mr 48,000 and 24,000 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.
Heidlas
J
J
Technische Universität Berlin, Fachbereich Lebensmitteltechnologie und Biotechnologie, Fachgebiet Chemisch-technische Analyse.
Tressl
R
R
D000094:Acetoin Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D002074:Butanones / Q000378:metabolism*; D002846:Chromatography, Affinity; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D003931:Diacetyl / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010422:Pentanones / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D002074:Butanones; D007659:Ketones; D010422:Pentanones; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D000094:Acetoin Dehydrogenase; D003931:Diacetyl