Conversion of vestitone to medicarpin in alfalfa (Medicago sativa L.) is catalyzed by two independent enzymes. Identification, purification, and characterization of vestitone reductase and 7,2'-dihydroxy-4'-methoxyisoflavanol dehydratase

J Biol Chem. 1994 Sep 2;269(35):22372-8.

Abstract

Pterocarpan phytoalexins are antimicrobial compounds in leguminous plants. The final step of pterocarpan biosynthesis, conversion of vestitone to medicarpin, was thought to be catalyzed by a single enzyme "pterocarpan synthase." We have shown that the pterocarpan synthase activity observed in crude extracts of alfalfa suspension cell cultures is the sum of two independent enzymatic activities: vestitone reductase, which catalyzes the NADPH-dependent reduction of vestitone to 7,2'-dihydroxy-4'-methoxyisoflavanol (DMI), and DMI dehydratase, which catalyzes loss of water and closure of an ether ring to form medicarpin. The first enzyme, vestitone reductase, was purified 1,840-fold to homogeneity by a 5-step procedure. Purified vestitone reductase showed a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 38 kDa. The native molecular mass measured by gel filtration was shown to be 34 kDa, indicating that vestitone reductase is a monomer. Vestitone reductase has strict substrate stereo specificity for (3R)-vestitone with a Km value of 45 microM. The second enzyme, DMI dehydratase, was partially purified 962-fold. DMI dehydratase had a native molecular mass of 38 kDa as estimated by gel filtration and a Km value of 5 microM for DMI. Both enzymes have a temperature optimum of 30 degrees C and a pH optimum of 6.0. The discovery of vestitone reductase and DMI dehydratase will facilitate future genetic manipulation of pterocarpan biosynthesis.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Benzopyrans / metabolism*
  • Catalysis
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Chromones / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Hydro-Lyases / metabolism*
  • Isoflavones
  • Medicago sativa / metabolism*
  • Molecular Structure
  • Oxidoreductases / isolation & purification
  • Oxidoreductases / metabolism*
  • Oxidoreductases Acting on CH-CH Group Donors*
  • Pterocarpans*
  • Spectrometry, Mass, Fast Atom Bombardment
  • Spectrophotometry, Ultraviolet

Substances

  • Benzopyrans
  • Chromones
  • Isoflavones
  • Pterocarpans
  • vestitone
  • medicarpin
  • Oxidoreductases
  • vestitone reductase
  • Oxidoreductases Acting on CH-CH Group Donors
  • 7,2'-dihydroxy-4'-methoxyisoflavanol dehydratase
  • Hydro-Lyases