A Minimal Mutation Trajectory Separates Inverting and Retaining Mechanisms on the Same Sialidase Structural Scaffold

Abstract

The recently discovered sialidase AmGH181 from the human gut symbiont Akkermansia muciniphila is remarkably selective to the sialyl T-antigen, a glycan found on mucins and cancer cells. However, the molecular basis for specificity and catalytic mechanism remains unknown in this enzyme family. Combining mutagenesis, X-ray crystallography, and QM/MM metadynamics, we show that specificity is governed by a flexible tryptophan–histidine pair, forming a “sugar tang” that precisely positions the sialyl T-antigen for catalysis. Hydrolysis of the sialyl-T antigen proceeds via a single-step S_N2 reaction and a ^3,6B/³S_O → [^3,6B]^‡ → ^3,6B/³S_O → ²C₅ conformational itinerary of the sialyl unit at subsite –1, resulting in the inversion of anomeric configuration. Remarkably, we altered the reaction stereochemistry by a double mutation, introducing a tyrosine to perform nucleophilic attack on the C2 of the sialyl unit at subsite –1, assisted by acid catalysis to promote aglycone departure. This retaining mutant acquired trans-sialidase activity and performed synthesis of 3′-sialyllactose in remarkably high yields. These findings suggest that the evolutionary divergence of retaining and inverting sialidases hinges on minimal active site mutations. The successful conversion of an inverting hydrolase into a retaining one provides an innovative strategy for harnessing inverting glycosidases for oligosaccharide synthesis via transglycosylation reactions.