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

The enzymatic hydrolysis of alpha-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 alpha-l-fucosidase unveiling a Michaelis (ES) complex in a C-1(4) (chair) conformation and a covalent glycosyl-enzyme intermediate in S-3(1) (skew-boat). First principles metadynamics simulations on isolated alpha-l-fucose strongly support a C-1(4)<-> H-3(4)<-> S-3(1) conformational itinerary for the glycosylation step of the reaction mechanism and indicate a strong "preactivation" of the C-1(4) complex to nucleophilic attack as reflected by free energy, C1-O1/05-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.