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)

Abstract

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 M_r of 37,500. The pH optimum of DMIRase was determined to be 7.8. The enzyme showed apparent Michaelis constants of 20 μm for 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavone and 58 μm 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.