Functional and Structural Characterization of a Flavonoid Glucoside 1,6-Glucosyltransferase from Catharanthus roseus

Sugarsugar glycosyltransferases play an important role in structural diversity of small molecule glycosides in higher plants. We isolated a cDNA clone encoding a sugarsugar glucosyltransferase (CaUGT3) catalyzing 1,6-glucosylation of flavonol and flavone glucosides for the first time from Catharanthus roseus. CaUGT3 exhibited a unique glucosyl chain elongation activity forming not only gentiobioside but also gentiotrioside and gentiotetroside in a sequential manner. We investigated the functional properties of CaUGT3 using homology modeling and site-directed mutagenesis, and identified amino acids positioned in the acceptor-binding pocket as crucial for providing enough space to accommodate flavonoid glucosides instead of flavonoid aglycones. These results provide basic information for understanding and engineering the catalytic functions of sugarsugar glycosyltransferases involved in biosynthesis of plant glycosides.