Analysis of the Reaction Coordinate of α-l-Fucosidases: A Combined Structural and Quantum Mechanical Approach

The enzymatic hydrolysis of α-l-fucosides is of importance in cancer, bacterial infections, and fucosidosis, a neurodegenerative lysosomal storage disorder. Here we show a series of snapshots along the reaction coordinate of a glycoside hydrolase family GH29 α-l-fucosidase unveiling a Michaelis (ES) complex in a 1C4 (chair) conformation and a covalent glycosyl-enzyme intermediate in 3S1 (skew-boat). First principles metadynamics simulations on isolated α-l-fucose strongly support a 1C4↔3H4↔3S1 conformational itinerary for the glycosylation step of the reaction mechanism and indicate a strong "preactivation" of the 1C4 complex to nucleophilic attack as reflected by free energy, C1−O1/O5−C1 bond length elongation/reduction, C1−O1 bond orientation, and positive charge development around the anomeric carbon. Analysis of an imino sugar inhibitor is consistent with tight binding of a chair-conformed charged species.