Affinity Chromatography, Substrate/Product Specificity, and Amino Acid Sequence Analysis of an IsoflavoneO-Methyltransferase from Alfalfa (Medicago sativaL.)

IsoflavoneO-methyltransferase (IOMT) is a key enzyme in the biosynthesis of the phytoalexin medicarpin in alfalfa.In vivo,the B-ring 4′-hydroxyl group of the isoflavone daidzein is methylated. Surprisingly, theO-methyltransferase activity measuredin vitropreferentially methylates the A-ring 7-hydroxyl group, a reaction that probably does not occurin vivo.To resolve this anomaly, we are attempting to clone the alfalfa IOMT. A substrate-based affinity chromatographic system was developed to purify the enzyme (molecular weight 41 kDa) to near homogeneity. Four internal peptide sequences were obtained from the purified protein, one of which has high (72%) sequence identity to a region of a catecholO-methyltransferase from barley. All four internal peptides, respectively, have about 55% amino acid sequence identity to four regions of 6α-hydroxymaackiain 3-O-methyltransferase fromPisum sativum,but have no sequence identity to alfalfa caffeic acid 3-O-methyltransferase 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.