ISOLATION AND CHARACTERIZATION OF A UDPGLUCOSE-FLAVONOL O-3-GLUCOSYLTRANSFERASE FROM ILLUMINATED RED CABBAGE (BRASSICA-OLERACEA CV RED DANISH) SEEDLINGS

A UDPGIc:flavonol O3-glucosyltransferase (EC 2.4.1.91) that catalyzes the formation of quercetin and kaempferol O3-gluco-sides has been purified about 1450-fold from illuminated red cabbage (Brassica oleracea cv Red Danish) seedlings with a 3.3% yield. Purification of the enzyme was achieved by (NH4)2SO4-precipitation, gel-filtration, ion-exchange chromatography on DEAE-Bio-Gel and Q-Sepharose, chromatofocusing, and electrophoresis in nondenaturing polyacrylamide (10%) gels. The enzyme preparation had a pH optimum between 5.8 and 6.2, isoelectric point in the pH range 4.25 to 4.55, a M(r) of 59,000, and it was composed of two similar subunits of M(r) 29,500. The glucosyltransferase reached half substrate saturation at 180 micromolar (UDPGlc) and 7 micromolar (quercetin) concentrations. Kaempferol, which was glucosylated at a relative rate of 87%, had a lesser affinity for the enzyme (K(m) approximately 12 micromolar). Flavanones, flavanols, flavones, dihydroflavonols, and anthocyanidins were not readily utilized as substrates, suggesting that the enzyme is specific for flavonol glucoside biosynthesis.