Structural and mechanistic investigations of protein S-glycosyltransferases.

Attachment of sugars to nitrogen and oxygen in peptides is ubiquitous in biology, but glycosylation of sulfur atoms has only been recently described. Here, we characterize two S-glycosyltransferases SunS and ThuS that selectively glycosylate one of five Cys residues in their substrate peptides; substitution of this Cys with Ser results in a strong decrease in glycosylation activity. Crystal structures of SunS and ThuS in complex with UDP-glucose or a derivative reveal an unusual architecture in which a glycosyltransferase type A (GTA) fold is decorated with additional domains to support homodimerization. Dimer formation creates an extended cavity for the substrate peptide, drawing functional analogy with O-glycosyltransferases involved in cell wall biosynthesis. This extended cavity contains a sharp bend that may explain the site selectivity of the glycosylation because the target Cys is in a Gly-rich stretch that can accommodate the bend. These studies establish a molecular framework for understanding the unusual S-glycosyltransferases.

The glycosyltransferase involved in thurandacin biosynthesis catalyzes both O- and S-glycosylation.

The S-glycosyltransferase SunS is a recently discovered enzyme that selectively catalyzes the conjugation of carbohydrates to the cysteine thiol of proteins. This study reports the discovery of a second S-glycosyltransferase, ThuS, and shows that ThuS catalyzes both S-glycosylation of the thiol of cysteine and O-glycosylation of the hydroxyl group of serine in peptide substrates. ThuS-catalyzed S-glycosylation is more efficient than O-glycosylation, and the enzyme demonstrates high tolerance with respect to both nucleotide sugars and peptide substrates. The biosynthesis of the putative products of the thuS gene cluster was reconstituted in vitro, and the resulting S-glycosylated peptides thurandacin A and B exhibit highly selective antimicrobial activity toward Bacillus thuringiensis.