Affinity chromatography, substrate/product specificity, and amino acid sequence analysis of an isoflavone O-methyltransferase from alfalfa (Medicago sativa L)

被引:29
作者
He, XZ [1 ]
Dixon, RA [1 ]
机构
[1] SAMUEL ROBERTS NOBLE FDN INC,DIV PLANT BIOL,ARDMORE,OK 73402
关键词
isoflavonoid biosynthesis; phytoalexins; cell culture; metabolic channeling;
D O I
10.1006/abbi.1996.0539
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Isoflavone O-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, the O-methyltransferase activity measured in vitro preferentially methylates 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 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 catechol O-methyltransferase hom barley. All four internal peptides, respectively, have about 55% amino acid sequence identity to four regions of 6 alpha-hydroxymaackiain 3-O-methyltransferase from Pisum 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. (C) 1996 Academic Press, Inc.
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页码:121 / 129
页数:9
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