Detection and characterization of anS-adenosyl-l-methionine: Flavonoid 7-O-methyltransferase from leaves ofPrunus x yedoensis matsum. with special reference to prunetrin formation

Abstract

An enzyme,S-adenosyl-l-methionine: flavonoid 7-O-methyltransferase (F7OMT), catalyzing the transfer of the methyl group fromS-adenosyl-l-methionine (SAM) to the 7 position of sophoricoside (5, 7, 4′-trihydroxyisoflavone 4′-O-glucoside) and some of the other flavonoids, was detected in extracts from leaves ofPrunus x yedoensis, and it was partially purified (about 203-fold) by a combination of gel filtration and ion-exchange column chromatographies. F7OMT was isolated as a soluble enzyme with a pH optimun of 7.5 in K-phosphate buffer. The molecular mass of F7OMT, which had an isoelectric point at pH 4.1, was estimated by elution from a column of Sephadex G-100 to be about 36 kDa. The activity of F7OMT was stimulated by 14 mM 2-Co2+ and reagents that react with sulfhydryl groups. The apparentKm values for sophoricoside, its aglycone genistein (5, 7, 4′-trihydroxyisoflavone) and quercetin were 1.49, 2.19 and 1.89 μM, respectively. The apparentKm value for SAM as methyl donor was 2.08 mM. The specificity of F7OMT for methyl acceptors was not strict; flavonols, flavanones and flavanonols in addition to isoflavones served as methyl acceptor. An examination ofP. x yedoensis leaves during spring and autumn showed variations in the activities of F7OMT and UDP-glucose: isoflavone 4′-O-glucosyltransferase (I4′ GT). The activities of F7OMT and I4′GT increased in enlarging leaf tissues and then markedly declined when the leaves approached maturation. In autumn leaves F7OMT activity was scarcely detected, but a small peak of I4′GT activity was observed during autumnal reddening.