Phytoalexin Synthesis in Soybean: Purification and Characterization of NADPH:2'-hydroxydaidzein Oxidoreductase From Elicitor-Challenged Soybean Cell Cultures

Arch Biochem Biophys. 1990 Feb 1;276(2):390-5. doi: 10.1016/0003-9861(90)90737-j.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cells, Cultured
  • Chromatography, Affinity
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Kinetics
  • Molecular Weight
  • Oxygenases / isolation & purification*
  • Oxygenases / metabolism
  • Plant Extracts / metabolism*
  • Sesquiterpenes
  • Soybeans / enzymology*
  • Substrate Specificity
  • Terpenes

Substances

  • Plant Extracts
  • Sesquiterpenes
  • Terpenes
  • phytoalexins
  • Oxygenases
  • isoflavone synthase