AFFINITY-CHROMATOGRAPHY, SUBSTRATE PRODUCT SPECIFICITY, AND AMINO-ACID-SEQUENCE ANALYSIS OF AN ISOFLAVONE O-METHYLTRANSFERASE FROM ALFALFA (MEDICAGO-SATIVA L)/

Isoflavone O-methyltransferase (IOMT) is a key enzyme in the biosynthe sis of the phytoalexin medicarpin in alfalfa. In vivo, the B-ring 4'-h ydroxyl group of the isoflavone daidzein is methylated. Surprisingly, the O-methyltransferase activity measured in vitro preferentially meth ylates the A-ring 7-hydroxyl group, a reaction that probably does not occur in vivo. To resolve this anomaly, we are attempting to clone the alfalfa IOMT. A substrate-based affinity chromatographic system was d eveloped to purify the enzyme (molecular weight 41 kDa) to near homoge neity. Four internal peptide sequences were obtained from the purified protein, one of which has high (72%) sequence identity to a region of a catechol O-methyltransferase hom barley. All four internal peptides , respectively, have about 55% amino acid sequence identity to four re gions of 6 alpha-hydroxymaackiain 3-O-methyltransferase from Pisum sat ivum, but have no sequence identity to alfalfa caffeic acid 3-O-methyl transferase or chalcone 2'-O-methyltransferase. The purified IOMT has substrate specificity toward isoflavones with a free 7-hydroxyl group, but can also methylate the 5-hydroxyl group of genistein. (C) 1996 Ac ademic Press, Inc.