Phytoalexin synthesis in soybean: purification and characterization of NADPH:2'-hydroxydaidzein oxidoreductase from elicitor-challenged soybean cell cultures.
An NADPH:2'-hydroxydaidzein oxidoreductase (HDR) from elicitor-challenged soybean cell cultures was purified to apparent homogeneity by a five-step procedure. The purification procedure included affinity adsorption on Blue Sepharose and elution of the enzyme with NADP+. It was shown by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis that HDR consists of only one polypeptide, which has a Mr about 34,700. The pH optimum of the reaction was 7.0. Apparent Michaelis constants determined for 2'-hydroxydaidzein, 2'-hydroxyformononetin, and NADPH were, respectively, 50, 60, and 56 microM. A low conversion of 2'-hydroxygenistein to the corresponding isoflavanone was also observed but isoflavones lacking a 2'-hydroxyl group and various other flavonoids did not serve as substrates. Enzymatically derived 2'-hydroxydihydrodaidzein gave a positive CD spectrum at 328 nm, which shows its 3R stereochemistry. Antibodies against HDR were raised in rats.
Fischer
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Lehrstuhl für Biochemie der Pflanzen, Biologisches Institute II, Universität Freiburg, Federal Republic of Germany.
Ebenau-Jehle
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C
Grisebach
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H
D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D010936:Plant Extracts / Q000378:metabolism*; D012717:Sesquiterpenes; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D013729:Terpenes; D000094162:Phytoalexins
D010936:Plant Extracts; D012717:Sesquiterpenes; D013729:Terpenes; D010105:Oxygenases; C048489:isoflavone synthase; D000094162:Phytoalexins