Publications

• Molecular cloning and expression of alfalfa (Medicago sativa L.) vestitone reductase, the penultimate enzyme in medicarpin biosynthesis.

  Guo L., Paiva N.L.

  Medicarpin, the major phytoalexin in alfalfa, is synthesized by way of the isoflavonoid branch of phenylpropanoid metabolism. One of the final steps of medicarpin biosynthesis, from vestitone to 7,2'-dihydroxy-4'-methoxyisoflavanol, is catalyzed by vestitone reductase. A 1245-bp cDNA clone which e ... >> More

  Medicarpin, the major phytoalexin in alfalfa, is synthesized by way of the isoflavonoid branch of phenylpropanoid metabolism. One of the final steps of medicarpin biosynthesis, from vestitone to 7,2'-dihydroxy-4'-methoxyisoflavanol, is catalyzed by vestitone reductase. A 1245-bp cDNA clone which encodes vestitone reductase was identified utilizing internal amino acid sequence of purified vestitone reductase. When expressed in Escherichia coli, the cloned enzyme exhibits strict substrate stereospecificity for (3R)-vestitone, as was observed for vestitone reductase purified from alfalfa. The calculated molecular weight of the protein (35,918) is similar to that of purified vestitone reductase from alfalfa (38 kDa by SDS-PAGE). The levels of vestitone reductase transcript (1.35 kb) greatly increase within 2 h of elicitor addition to alfalfa cell suspension cultures, preceding the rapid increases in vestitione reductase enzyme activity and medicarpin biosynthesis. In healthy alfalfa plants, the highest levels of transcripts were detected in roots and root nodules, consistent with the synthesis of medicarpin and its conjugate in these tissues. The cloning of the vestitone reductase gene provides a specific tool for the study and manipulation of pterocarpan biosynthesis in legumes. << Less

  Arch. Biochem. Biophys. 320:353-360(1995) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The 'pterocarpan synthase' of alfalfa: association and co-induction of vestitone reductase and 7,2'-dihydroxy-4'-methoxy-isoflavanol (DMI) dehydratase, the two final enzymes in medicarpin biosynthesis.

  Guo L., Dixon R.A., Paiva N.L.

  Vestitone reductase and 7,2'-dihydroxy-4'-methoxy-isoflavanol (DMI) dehydratase are the two final enzymes in medicarpin biosynthesis in alfalfa (Medicago sativa). Although two independent enzymes, vestitone reductase and DMI dehydratase can be loosely associated in low ionic strength buffers, pres ... >> More

  Vestitone reductase and 7,2'-dihydroxy-4'-methoxy-isoflavanol (DMI) dehydratase are the two final enzymes in medicarpin biosynthesis in alfalfa (Medicago sativa). Although two independent enzymes, vestitone reductase and DMI dehydratase can be loosely associated in low ionic strength buffers, presumably by a weak protein-protein interaction. The activities of vestitone reductase and DMI dehydratase increased approximately 3-fold 6 hours after elicitor treatment in alfalfa suspension cell culture. The activities remained at maximal levels for 40 hours, correlating with a steady increase in the medicarpin content of the cells. Medicarpin produced in vitro from vestitone by the action of vestitone reductase and DMI dehydratase was found to be (-)-medicarpin (6aR,11aR-medicarpin), possessing the same stereochemistry as medicarpin produced in vivo. << Less

  FEBS Lett. 356:221-225(1994) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• 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.

  Guo L., Dixon R.A., Paiva N.L.

  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 crud ... >> More

  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. << Less

  J. Biol. Chem. 269:22372-22378(1994) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.