Purification and characterization of two enone reductases from Saccharomyces cerevisiae.
Two enone reductases catalyzing irreversibly the enantioselective reduction of alpha,beta-unsaturated carbonyls have been purified 165-fold and 257-fold, respectively, from the cytosolic fraction of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, blue Sepharose CL-6B chromatography and Superdex 200 preparation-grade filtration. One enzyme (E I) was NADPH-dependent, showed a molecular mass of 75 kDa and decomposed under denaturing electrophoretic conditions into two subunits of 34 kDa and 37 kDa. The other enzyme (E II) was NADH linked and the molecular mass estimated by means of Superdex 200 preparation-grade filtration, was 130 kDa. The enzyme decomposed into subunits of 56 kDa and 64 kDa on SDS/PAGE. Both enzymes were most active at pH 4.8 and accepted 1-octen-3-one, 1-hexen-3-one, 3-alken-2-ones, 4-alken-3-ones, 2-cyclohexen-1-ones, 2-alkenals, 2,4-alkadienals, 2-phenyl-2-alkenals, and 2-alkyl-2-alkenals as substrates. Both enzymes showed their highest activities with 1-hexen-3-one and 1-octen-3-one, respectively. The Km values for 1-octen-3-one were estimated as 0.54 mM (E I) and 0.20 mM (E II), respectively. Both enzymes catalyzed the enantioselective reduction of cis- and trans-2-phenyl-2-butenal into (R)-2-phenylbutanal.
Wanner
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Technische Universität Berlin, Institut für Biotechnologie, Fachgebiet Chemisch-technische Analyse, Germany.
Tressl
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D000447:Aldehydes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D000442:Octanols / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D000447:Aldehydes; D007659:Ketones; D000442:Octanols; D009243:NAD; C057348:2-octenal; D009249:NADP; C017582:3-octanone; C113805:1-octen-3-one; D010088:Oxidoreductases