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.
Guo
L
L
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73402.
Dixon
R A
RA
Paiva
N L
NL
D001578:Benzopyrans / Q000378:metabolism*; D002384:Catalysis; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002867:Chromones / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006836:Hydro-Lyases / Q000378:metabolism*; D007529:Isoflavones; D000455:Medicago sativa / Q000378:metabolism*; D015394:Molecular Structure; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D036343:Pterocarpans; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013056:Spectrophotometry, Ultraviolet; D000094162:Phytoalexins
D001578:Benzopyrans; D002867:Chromones; D006836:Hydro-Lyases; D007529:Isoflavones; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D036343:Pterocarpans; C089075:vestitone; C047353:medicarpin; D000094162:Phytoalexins; C071072:vestitone reductase; C089074:7,2'-dihydroxy-4'-methoxyisoflavanol dehydratase