MOLECULAR-CLONING AND EXPRESSION OF ALFALFA (MEDICAGO-SATIVA L) VESTITONE REDUCTASE, THE PENULTIMATE ENZYME IN MEDICARPIN BIOSYNTHESIS

Medicarpin, the major phytoalexin in alfalfa, is synthesized by way of the isoflavonoid branch of phenylpropanoid metabolism, One of the fin al 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 exp ressed in Escherichia coli, the cloned enzyme exhibits strict substrat e stereospecificity for (3R)-vestitone, as was observed for vestitone reductase purified from alfalfa. The calculated molecular weight of th e protein (35,918) is similar to that of purified vestitone reductase from alfalfa (38 kDa by SDS-PAGE). The levels of vestitone reductase t ranscript (1.35 kb) greatly increase within 2 h of elicitor addition t o alfalfa cell suspension cultures, preceeding the rapid increases in vestitone reductase enzyme activity and medicarpin biosynthesis, In he althy 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 redu ctase gene provides a specific tool for the study and manipulation of pterocarpan biosynthesis in legumes. (C) 1995 Academic Press, Inc.