Isolation and characterization of a UDP-glucose:cyanidin 3-O-glucosyltransferase from grape cell suspension cultures (Vitis vinifera L)

An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 3-hydroxyl group of cyanidin has been isolated from a Vitis vinifera cell suspension culture. The enzyme was purified 75-fold by ion-exchange, chromatofocusing and gel filtration liquid chromatography with a recovery of 3.8%. The enzyme had a pH optimum at 8.0, The glucosyltransferase showed the highest activity with cyanidin as acceptor but also utilized delphinidin to a significant degree, Pelargonidin, peonidin and malvidin were glucosylated but at a lower rate. An apparent K-m value of 1.2 mM for UDP-glucose was determined. At an equal concentration of UDP-glucose the apparent K-m values of the enzyme for the accepters were 18 mu M for cyanidin (3', 4' OH) and 28 mu M for delphinidin (3', 4', 5' OH), The purified enzyme had an isoelectric point of 4.55 and a molecular weight of 56 kDa. Substrate affinity of the enzyme indicates that it is the first step of two anthocyanin branches which begin with cyanidin 3-glucoside and delphinidin 3-glucoside. Also, the lower activity of the enzyme with 3'- and 5'-O-methylated derivatives of cyanidin and delphinidin would explain in part why methylation occurs after the glucosylation step.