Shaping the Glycan Landscape: Hidden Relationships between Linkage and Ring Distortions Induced by Carbohydrate-Active Enzymes

Carbohydrate-active enzymes (CAZymes) catalyze glycan remodeling by forming and cleaving glycosidic bonds. An often-observed aspect of their catalytic mechanisms, particularly in glycosidases, is monosaccharide ring distortion that brings the substrate from a stable solution conformation to a reactive state. To what extent this distortion is promoted by steric constraints in the enzyme's binding pocket or by more elusive dynamical effects associated with the glycan conformational flexibility is still a matter of debate. In our work, we quantify the conformational phase-space changes experienced by glycans upon CAZyme binding by means of enhanced-sampling molecular dynamics simulations. Our results reveal a novel correlation between torsional degrees of freedom along the glycosidic bonds and the pucker degrees of freedom within the mannose ring at the -1 subsite of glycan M5G0 upon binding to the Golgi α-mannosidase II enzyme. Key factors driving this torsional phase-space reshaping and the associated transition from solution ⁴<i>C</i>₁ to ^O<i>S</i>₂/<i>B</i>_2,5 reactive pucker states include tight interactions with a protonated aspartic acid and a Zn²⁺ ion in the catalytic site. Comparative studies with ER α-mannosidase I show a different mechanism, where torsional conformations and ring distortion of the M9 glycan substrate are not correlated. By validating against previous computational and experimental studies, we theoretically predict the influence of amino acid mutations and altered glycan compositions on the conformational transition mechanisms. Our findings provide new insights into CAZyme specificity and effectiveness, laying the groundwork for the design of selective inhibitors targeting glycosylation-related diseases.