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

Abstract

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 acceptors were 18 μM for cyanidin (3′, 4′ OH) and 28 μM 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.