Open AccessPurification and characterization of two enone reductases from Saccharomyces cerevisiae
Author(s) -
Wanner Peter,
Tressl Roland
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2550271.x
Subject(s) - sephadex , size exclusion chromatography , chemistry , enzyme , molecular mass , sepharose , chromatography , saccharomyces cerevisiae , enantioselective synthesis , biochemistry , stereochemistry , yeast , catalysis
Two enone reductases catalyzing irreversibly the enantioselective reduction of α,β‐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 K m 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.