Purification and Properties of Flavonol-Ring B Glucosyltransferase from Chrysosplenium americanum

Abstract

A novel glucosyltransferase which catalyzed the transfer of glucose from UDP-glucose to positions 2'' and 5'' of partially methylated flavonols was isolated from the shoots of C. americanum Schwein ex Hooker. It was purified 225-fold by ammonium sulfate precipitation and successive chromatogrpahy on Sephadex G-100, hydroxyapatite and polybuffer ion exchanger. This glucosyltransferase appeared to be a single polypeptide with an apparent MW of 42,000 daltons, pH optimum of 7.5-8.0, and an isoelectric point of 5.1. It had low but similar Km values for the 2'' and 5'' positions of flavonol substrates and the cosubstrate UDP-glucose and was inhibited by both reaction products, the glucosides formed and UDP. Glucosyltransferase activity was independent of divalent cations, was not inhibited by EDTA, but showed requirement for SH groups. The differential effect on enzyme activity of metal ions, especially cupric ion, and various SH group reagents seemed to indicate the involvement of 2 active sites in the glucosylation reaction; the site specific for 2'' activity being more susceptible than that of the 5'' activity. The substrate specificity expressed by this glucosyltransferase and the requirement of at least 2 para-oriented B-ring substituents (at 2'' and 5'') for activity support this view.