S-glycosyltransferase UGT74B1 can glycosylate both S- and O-acceptors: mechanistic insights through substrate specificity

Abstract

UGT74B1 from Arabidopsis thaliana is one of the few characterized glycosyltransferases able to generate a thioglycosidic linkage in vivo, using the sulfur atom of thiohydroximate as the nucleophile in the glycosylation reaction. This critical biosynthetic step in glucosinolate production has been documented. Yet little is known about the molecular mechanism that enables this rare and unusual glycosylation at the sulfur atom. To identify the role of this atom in the glycosylation reaction and unravel the mechanism of UGT74B1, we used a range of substrates containing either sulfur or oxygen. We first demonstrated that the enzyme could catalyze the glycosylation of thiohydroximates but also of O-containing hydroximates analogs. If KM values were shown to be close between analogs, the reaction catalytic rate kcat was 50–100 lower in the case of hydroximates. The glycosylation reaction is catalyzed through deprotonation of the acceptor, which was confirmed by the removal of catalytic carboxylic residues by site-directed mutagenesis. Moreover, using a range of simple phenols and thiophenols as UGT74B1 substrate acceptors for glycosylation, we concluded that the glycosylation reaction rate is correlated to the acceptor atom acidity, and not to the nature of this nucleophilic atom (oxygen or sulfur).