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.
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.[1]References
- 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. J. Biol. Chem. (1994) [Pubmed]
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