CONVERSION OF VESTITONE TO MEDICARPIN IN ALFALFA (MEDICAGO-SATIVA L) IS CATALYZED BY 2 INDEPENDENT ENZYMES - IDENTIFICATION, PURIFICATION, AND CHARACTERIZATION OF VESTITONE REDUCTASE AND 7,2'-DIHYDROXY-4'-METHOXYISOFLAVANOL DEHYDRATASE

Pterocarpan phytoalexins are antimicrobial compounds in leguminous pla nts. The final step of pterocarpan biosynthesis, conversion of vestito ne to medicarpin, was thought to be catalyzed by a single enzyme ''pte rocarpan synthase.'' We have shown that the pterocarpan synthase activ ity 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'-dih ydroxy-4'-methoxyisoflavanol (DMI), and DMI dehydratase, which catalyz es loss of water and closure of an ether ring to form medicarpin. The first enzyme, vestitone reductase, was purified 1,840-fold to homogene ity by a 5-step procedure. Purified vestitone reductase showed a singl e band on SDS-polyacrylamide gel electrophoresis with an estimated mol ecular mass of 38 kDa. The native molecular mass measured by gel filtr ation 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 K-m value of 45 mu M. The second enzyme, DMI dehydratase, was partially purified 962-fold. DMI dehydratase had a n ative molecular mass of 38 kDa as estimated by gel filtration and a K- m value of 5 mu M for DMI. Both enzymes have a temperature optimum of 30 degrees C and a pH optimum of 6.0. The discovery of vestitone reduc tase and DMI dehydratase will facilitate future genetic manipulation o f pterocarpan biosynthesis.