Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures.
Two isoenzymes of an NADP+ -dependent cinnamyl alcohol dehydrogenase and an NAD+ - dependent aliphatic alcohol dehydrogenase were extracted from cell suspension cultures of soybean (Glycine max L., var. Mandarin) which form lignin during growth. These enzymes could be separated from each other by chromatography on DEAE-cellulose and hydroxyapatite. The cinnamyl alcohol dehydrogenase isoenzymes were partially purified by (NH4)2SO4 fractionation, and column chromatography on DEAE-cellulose, Sephadex G-100, and hydroxyapatite. The molecular weight of the enzymes were estimated by the elution volumes from a Sephadex G-100 column and were found to be about 43,000 (isoenzyme 1) and 69,000 (isoenzyme 2). Maximum rates of reaction were observed in the case of coniferyl alcohol oxidation at pH 9.2 (Isoenzyme 1) and pH 8.8 (isoenzyme 2); in the reverse reaction pH 6.5 was optimal for isoenzyme 2. Whereas isoenzyme 1 is specific for coniferyl alcohol, isoenzyme 2 can also oxidize cinnamyl alcohol and a number of substituted cinnamyl alcohols, Km values for substituted cinnamaldehydes are 3-11 times lower than for the corresponding alcohols. Neither isoenzyme reacted with benzyl alcohol, anisic alcohol or ethanol. Substrate inhibition for the forward and reverse reaction was found with isoenzyme 2 but not with isoenzyme 1. The equilibrium constant was determined to be about 10(9) in favour of coniferaldehyde reduction. The possible role of the cinnamyl alcohol dehydrogenase in lignin biosynthesis is discussed.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D002934:Cinnamates; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD; D010944:Plants / Q000201:enzymology*; D013025:Glycine max; D013329:Structure-Activity Relationship
D002934:Cinnamates; D007527:Isoenzymes; D009243:NAD; D000429:Alcohol Oxidoreductases