AFFINITY-CHROMATOGRAPHY, SUBSTRATE PRODUCT SPECIFICITY, AND AMINO-ACID-SEQUENCE ANALYSIS OF AN ISOFLAVONE O-METHYLTRANSFERASE FROM ALFALFA (MEDICAGO-SATIVA L)/
Xz. He et Ra. Dixon, AFFINITY-CHROMATOGRAPHY, SUBSTRATE PRODUCT SPECIFICITY, AND AMINO-ACID-SEQUENCE ANALYSIS OF AN ISOFLAVONE O-METHYLTRANSFERASE FROM ALFALFA (MEDICAGO-SATIVA L)/, Archives of biochemistry and biophysics, 336(1), 1996, pp. 121-129
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.