Sagiroglugil, M.; Castillo, B. P.; Leanca, M. A.; Kitano, E. S.; Mycroft-West, C. J.; Papadakis, G.; Gafitescu, M. A.; Mohammed, S.; Davis, B. G.; Rovira, C.; Wu, L.

Bifunctional glycosyltransferases underpin biosynthesis of essential glycans, including bacterial capsular antigens, and eukaryotic glycosaminoglycans. Typically, these enzymes transfer hexose sugars from activated nucleotide-phosphate donors, forming new glycosidic bonds with either retention or inversion of anomeric stereochemistry. Retaining hexosyl transfer typically proceeds via non-covalent SNi-type pathways, but recent studies of octulosyl Kdo-transferases have reignited debate over covalent mechanisms. Here we comprehensively dissected the activity of the archetypal bifunctional glycosyltransferase PmHS2, providing X-ray structures of ligand complexes alongside atomistic simulations to delineate full substrate-to-product reaction trajectories. Combined computational, biochemical and evolutionary evidence suggests that PmHS2 retaining GlcNAc-transfer likely proceeds via a previously undescribed covalent mechanism, involving considerable assistance from departing nucleotide-phosphate rotation. In mapping reaction pathways, we also report the first example of a glycosyl donor substrate coopted as an acceptor, which enables enzymatic self-priming for polymerization. These new covalent and self-priming functionalities highlight unexplored routes to modulate or inhibit polysaccharide biosynthesis.