Conformation-dependent donor selectivity in the xanthan gum glycosyltransferase GumK revealed by AI-based docking

The interdomain flexibility of GT-B fold glycosyltransferases regulates substrate binding and catalysis, yet the role of local structural variations in donor substrate specificity remains unclear. GumK, a GT70 enzyme from Xanthomonas campestris, exhibits local plasticity within its donor-binding pocket. We classify this plasticity into two conformational states, defined by the presence (closed state) or absence (open state) of a conserved hydrophobic interaction stabilizing the pocket. Using the AI-enhanced docking approach GNINA, we investigated the relationship between these states and substrate specificity by comparing UDP-glucuronate with five acidic and neutral ligand analogs. While docking scores showed limited discrimination among ligands, distance-based analysis between the sugar C6 atom and Lys307 revealed conformation-dependent trends. In the open state, negatively charged sugars preferentially interact with Lys307 via their carboxylate groups. Conversely, the closed state favors interactions with the pyrophosphate moiety. To provide orthogonal validation, we generated ensembles of GumK-substrate complexes using AI-based cofolding methods (AlphaFold3 and Boltz). While Boltz-2 affinities correctly ranked substrate specificity, only the GNINA-generated ensembles were consistent with physics-based simulations in capturing the conformational basis of selectivity. These results suggest that donor specificity arises from the interplay between ligand chemistry and binding-site plasticity rather than from a single rigid binding mode, and highlight complementary strengths of docking and cofolding approaches for studying flexible CAZymes.