Plant fucosyltransferase FUT11 distorts the sugar acceptor to catalyze via a transient oxocarbenium intermediate mechanism

Glycosyltransferases catalyze glycosidic bond formation by activating the donor sugar, while the sugar acceptor substrate is considered passive, maintaining a chair conformation during catalysis. We challenge this through a multidisciplinary study of Arabidopsis thaliana FUT11, a core α1,3-fucosyltransferase essential for plant development and reproduction. AtFUT11 adopts a GT-B fold with an additional N-terminal subdomain that anchors the G0 N-glycan, while the α1,3 arm is mainly recognized by the acceptor Rossmann subdomain. The α1,6 arm remains solvent-exposed, allowing diverse modifications, while solvent exposure of the central mannose’s OH2 explains tolerance for β1,2-xylose. Remarkably, simulations suggest the catalytic base Glu158 may promote the innermost GlcNAc’s transient puckering distortion to align the hydroxyl for nucleophilic attack. This enables an asynchronous SN2-like mechanism bordering SN1 character, with formation of a transient oxocarbenium ion triggered by pyrophosphate departure, followed by nucleophilic attack coupled with proton transfer. Homology with human FUT9 explains AtFUT11’s side activity on LacNAc, revealing plasticity and evolutionary convergence between plant and mammalian antenna-fucosyltransferases. Researchers mapped how the plant enzyme FUT11 adds fucose to N-glycans. Microarrays, crystal structures and simulations indicate the enzyme can distort the acceptor sugar during catalysis, informing glycoprotein engineering.