ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 336, No. 1, December 1, pp. 121–129, 1996 Article No. 0539 Affinity Chromatography, Substrate/Product Specificity, and Amino Acid Sequence Analysis of an Isoflavone O-Methyltransferase from Alfalfa ( Medicago sativa L.) 1 Xian-Zhi He and Richard A. Dixon 2 Plant Biology Division, Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, Oklahoma 73402 Received July 11, 1996, and in revised form September 11, 1996 derived from yeast cell walls (2). Medicarpin is synthesized Isoflavone O-methyltransferase (IOMT) is a key en- via the isoflavonoid branch of the phenylpropanoid path- zyme in the biosynthesis of the phytoalexin medicarpin way. Accumulation of medicarpin is preceded by large in- in alfalfa. In vivo, the B-ring 4-hydroxyl group of the creases in the extractable activities of all the known en- isoflavone daidzein is methylated. Surprisingly, the O- zymes involved in its formation from L-phenylalanine (2). methyltransferase activity measured in vitro preferen- Eight of the 13 genes required for the biosynthesis of the tially methylates the A-ring 7-hydroxyl group, a reac- intermediates leading to medicarpin have been cloned and tion that probably does not occur in vivo. To resolve well studied (3, 4). However, the nature of the enzymatic this anomaly, we are attempting to clone the alfalfa step resulting in methylation of the 9-position of medicar- IOMT. A substrate-based affinity chromatographic sys- pin (4-position, isoflavone numbering) is still unclear. tem was developed to purify the enzyme (molecular In a radiolabeled precursor study, 14 C-labeled formono- weight 41 kDa) to near homogeneity. Four internal pep- netin (7-hydroxy-4-methoxyisoflavone) was efficiently in- tide sequences were obtained from the purified protein, corporated into medicarpin in CuCl 2 -treated alfalfa seed- one of which has high (72%) sequence identity to a re- lings (5), suggesting that the methoxyl group at the 9- gion of a catechol O-methyltransferase from barley. All position of medicarpin originates from formononetin, four internal peptides, respectively, have about 55% which would itself be formed by methylation of the 4- amino acid sequence identity to four regions of 6a-hy- position of daidzein (7, 4-dihydroxyisoflavone). Further- droxymaackiain 3-O-methyltransferase from Pisum sat- more, a mutant of subterranean clover (Trifolium sub- ivum, but have no sequence identity to alfalfa caffeic terraneum), which had lost the ability to produce for- acid 3-O-methyltransferase or chalcone 2-O-methyl- mononetin, accumulated increased amounts of daidzein, transferase. The purified IOMT has substrate specific- suggesting that daidzein is the immediate precursor of ity toward isoflavones with a free 7-hydroxyl group, but formononetin (6). However, paradoxically, radiolabeled can also methylate the 5-hydroxyl group of genistein. daidzein is not incorporated into medicarpin in CuCl 2 -  1996 Academic Press, Inc. Key Words: isoflavonoid biosynthesis; phytoalexins; treated alfalfa seedlings (5). It was once suggested that cell culture; metabolic channeling. the methylation of the 4-position of isoflavones was an integral part of the aryl migration reaction occurring as part of the formation of isoflavone from its corresponding flavanone (7), but this now seems unlikely in view of the Alfalfa (Medicago sativa L.) rapidly accumulates the an- demonstration that the aryl migration is catalyzed by a timicrobial pterocarpan phytoalexin medicarpin upon fun- methyl group donor-independent cytochrome P450 en- gal infection, as an important component of its disease zyme (8). resistance response (1). Synthesis of this compound may It would be expected that an isoflavone 4-O-methyl- also be stimulated by treatment of seedlings with CuCl 2 transferase (4-IOMT) 3 would be responsible for the or exposure of cell suspension cultures to elicitor molecules 3 Abbreviations used: IOMT, isoflavone O-methyltransferase; 1 This work was supported by the Samuel Roberts Noble Founda- COMT, caffeic acid O-methyltransferase; ChalOMT, chalcone O- methyltransferase; DMSO, dimethyl sulfoxide; PVPP, polyvinyl- tion. 2 To whom correspondence should be addressed. Fax: (405) 221- polypyrrolidone; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl- L-homocysteine; FPLC, fast protein liquid chromatography. 7380. E-mail: radixon@noble.org. 121 0003-9861/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.