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 in diverse biological environments. Often, a key aspect of their catalytic mechanism is a monosaccharide chair-to-boat distortion that brings the substrate from a stable solution conformation to a reactive state. Using enhanced- sampling molecular dynamics simulations, we demonstrate that the ring distortion experienced by the glycan M5G0 upon binding to the Golgi α-mannosidase II (MII) enzyme actively correlates with a change of its global conformation. In solution, M5G0 adopts diverse conformers, all favoring the ⁴ C ₁ chair for the mannose at subsite -1 with respect to the bond cleavage point. Binding to MII narrows the glycan's phase space to only two low-energy conformers, which respectively correlate with the two distinct pucker states ⁴ C ₁ and ⁰ H ₅ . Key factors driving this phase-space reshaping include binding to specific amino acids and a Zn ²⁺ ion in the catalytic site. Comparative studies with ER α-mannosidase I show a different mechanism, where the enzyme enforces glycan conformations and ring distortion of the substrate independently. Our findings provide mechanistic insights into CAZyme specificity and effectiveness, laying the groundwork for the design of selective inhibitors targeting glycosylation-related diseases, including cancer.