Fast protein liquid chromatographic purification and some properties of a partially O-methylated flavonol glucoside 2′-/5′- O-methyltransferase

Abstract

A flavonol O-methyltransferase was partially purified from Chrysosplenium americanum by fractional precipitation with ammonium sulphate followed by gel filtration and chromatofocusing using an FPLC system. The enzyme which was purified 420-fold catalysed the transfer of the methyl group of SAM to the 2′- or 5′-positions of partially methylated flavonol glucosides, the two terminal methylation steps in the biosynthesis of Chrysosplenium flavonoids. The enzyme had a pH optimum of 7 in Pi buffer, a pI of < 5, an M, of 57 000, no Mg 2+ requirement and was inhibited by both N-ethylmaleimide and phenylmercuriacetate. The K m value for the flavonol substrate was 2 μM and that for SAM was 100 μM. The role of this enzyme is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue.