| dc.description |
A novel enzyme, UDP-D-galactose:flavonol 3-O-galactosyltransferase (F3GaT), catalyzing the transfer of D-galactose from UDP-D-galactose to the 3 position of 5,7,4′-trihydroxyflavonol (kaempferol), was detected in and purified about 404-fold from seedlings of Vigna mungo by precipitation with ammonium sulfate, chromatography on Sephadex G-100 and chromatofocusing. The enzyme was separated by this procedure from a coexisting UDP-D-glucose:flavonol 3-O-glucosyltransferase (F3GT), which was simultaneously purified about 189-fold. F3GaT was isolated as a soluble enzyme with pH optima of 8.0 in imidazole-HCl buffer and 7.5 in histidine-HCl buffer. F3GT had the same pH optima. The M<sub>r</sub> of both F3GaT and F3GT, which had isoelectric points of 5.1 and 6.1, respectively, was estimated by elution from a column of Sephadex G-100 to be about 43,000. The activities of F3GaT and F3GT were stimulated by 14 mM 2-mercaptoethanol and strongly inhibited by 1 mM Cu<sup>2+</sup>, 1 mM Zn<sup>2+</sup>, and various reagents that react with sulfhydryl groups. Among various possible substrates for F3GaT that were tested, kaempferol, isorhamnetin and quercetin were the best. The K<sub>m</sub> values for kaempferol and UDP-D-galactose were determined to be 0.40 μM and 125 μM, respectively. Similarly, F3GT had low K<sub>m</sub> values of 0.69 μM for kaempferol and 1.67 mM for UDP-D-glucose. F3GaT and F3GT mediated the transfer of galactose and glucose, respectively, to the 3-hydroxyl groups exclusively of kaempferol, isorhamnetin and quercetin. Rhamnetin also functioned as a galactosyl acceptor though less efficiently. |
