Year: 2026
Type: preprint
Abstract: 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 S N i -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.
Language: English
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Awards: G1105844351, G1272900390, G145641598, Rosalind Franklin Institute - Next Generation Chemistry, G2089336561, G491712853, G5635532810, Recurrent funding for the RFI 2019 to 2021, G593054264, G7139298319, G734693063, G7395345766, Investigating the molecular basis of Heparan Sulfate biosynthesis, G7805626310, G7863371703, RFI Phase 2, G8400896975, G8497050386, G8667377315, Investigating the molecular basis of Heparan Sulfate biosynthesis
OpenAlex ID: W7223150547