<i>O</i>-/<i>N</i>-/<i>S</i>-Specificity in Glycosyltransferase Catalysis: From Mechanistic Understanding to Engineering

Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds in carbohydrates and glycoconjugates, with various outcomes depending not only on acceptor molecules they bind but also type bond form (C–O, C–N, C–S, or C–C). Here we show that glucosyltransferase UGT1 from indigo plant Polygonum tinctorium catalyzes either N-, O-, S-glycosylation similar rates. We solve structure enzyme complex its donor substrates elucidate molecular basis S-specificities using experimental mutagenesis QM/MM simulations, revealing distinct mechanisms for S-glycosylation. active site can be engineered to increase favor one three glycosylation activities over another. These results will foster design more specific variants production glycosides.