Stereoisomerism in plant disease resistance: induction and isolation of the 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase, an enzyme introducing chirality during synthesis of isoflavonoid phytoalexins in pea (Pisum sativum L).
Treatment of pea seedlings with CuCl2 induced the activity of the enzyme NADPH:7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase (DMIRase) that introduces (+) stereoisomerism in pisatin. DMIRase was purified approximately 7000 fold from CuCl2-treated pea seedlings to apparent homogeneity by a six-step process. The purification sequence included (NH4)2SO4 fractionation, gel filtration on AcA 44, chromatography on DEAE-Bio-Gel,phenyl-Sepharose CL-4B, and Reactive Red 120-agarose, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration and denaturing electrophoresis showed that the enzyme consisted of a single polypeptide chain with an Mr of 37,500. The pH optimum of DMIRase was determined to be 7.8. The enzyme showed apparent Michaelis constants of 20 microM for 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone and 58 microM for NADPH. The reaction product of the enzyme, sophorol, gave a distinct negative Cotton effect in the region 300-360 nm, which indicated 3S configuration of the molecule. Antibodies against the enzyme were raised in rabbits and characterized for specificity.
Sun
Y J
YJ
Institute of Food Science, Cornell University, Geneva, New York 14456.
Wu
Q D
QD
Van Etten
H D
HD
Hrazdina
G
G
D002942:Circular Dichroism; D003300:Copper / Q000494:pharmacology; D004790:Enzyme Induction; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D010935:Plant Diseases; D010936:Plant Extracts / Q000096:biosynthesis*; D010946:Plants, Medicinal; D012717:Sesquiterpenes; D013379:Substrate Specificity; D013729:Terpenes; D000094162:Phytoalexins
D010936:Plant Extracts; D012717:Sesquiterpenes; D013729:Terpenes; D003300:Copper; D010088:Oxidoreductases; C066924:7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase; D009247:NADH, NADPH Oxidoreductases; C029892:cupric chloride; D000094162:Phytoalexins