Hexosyl transfer via a covalent intermediate within a dual retaining and inverting enzyme
- 1Rosalind Franklin Institute
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 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.
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BibTeX
@misc{sagiroglugil2026hexosyl,
title = {{Hexosyl transfer via a covalent intermediate within a dual retaining and inverting enzyme}},
author = {M. Sagiroglugil and B. P. Castillo and M. A. Leanca and E. S. Kitano and C. J. Mycroft-West and G. Papadakis and M. A. Gafitescu and S. Mohammed and B. G. Davis and C. Rovira and L. Wu},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.20.760405},
url = {https://www.biorxiv.org/content/10.64898/2026.10.20.760405v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Hexosyl transfer via a covalent intermediate within a dual retaining and inverting enzyme
AU - M. Sagiroglugil
AU - B. P. Castillo
AU - M. A. Leanca
AU - E. S. Kitano
AU - C. J. Mycroft-West
AU - G. Papadakis
AU - M. A. Gafitescu
AU - S. Mohammed
AU - B. G. Davis
AU - C. Rovira
AU - L. Wu
PY - 2026
DA - 2026/10/20
PB - bioRxiv
DO - 10.64898/2026.10.20.760405
UR - https://www.biorxiv.org/content/10.64898/2026.10.20.760405v1
AB - 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.
ER -