Publications

• 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.

• The missing link in leguminous pterocarpan biosynthesis is a dirigent domain-containing protein with isoflavanol dehydratase activity.

  Uchida K., Akashi T., Aoki T.

  Pterocarpan forms the basic structure of leguminous phytoalexins, and most of the isoflavonoid pathway genes encoding the enzymes responsible for its biosynthesis have been identified. However, the last step of pterocarpan biosynthesis is a ring closure reaction, and the enzyme that catalyzes this ... >> More

  Pterocarpan forms the basic structure of leguminous phytoalexins, and most of the isoflavonoid pathway genes encoding the enzymes responsible for its biosynthesis have been identified. However, the last step of pterocarpan biosynthesis is a ring closure reaction, and the enzyme that catalyzes this step, 2'-hydroxyisoflavanol 4,2'-dehydratase or pterocarpan synthase (PTS), remains as an unidentified 'missing link'. This last ring formation is assumed to be the key step in determining the stereochemistry of pterocarpans, which plays a role in their antimicrobial activity. In this study, a cDNA clone encoding PTS from Glycyrrhiza echinata (GePTS1) was identified through functional expression fractionation screening of a cDNA library, which requires no sequence information, and orthologs from soybean (GmPTS1) and Lotus japonicus (LjPTS1) were also identified. These proteins were heterologously expressed in Escherichia coli and biochemically characterized. Surprisingly, the proteins were found to include amino acid motifs characteristic of dirigent proteins, some of which control stereospecific phenoxy radical coupling in lignan biosynthesis. The stereospecificity of substrates and products was examined using four substrate stereoisomers with hydroxy and methoxy derivatives at C-4'. The results showed that the 4R configuration was essential for the PTS reaction, and (-)- and (+)-pterocarpans were produced depending on the stereochemistry at C-3. In suspension-cultured soybean cells, levels of the GmPTS1 transcript increased temporarily prior to the peak in phytoalexin accumulation, strongly supporting the possible involvement of PTS in pterocarpan biosynthesis. << Less

  Plant Cell Physiol. 58:398-408(2017) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.