Combined Inhibitor Free‐Energy Landscape and Structural Analysis Reports on the Mannosidase Conformational Coordinate

Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence‐based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X‐ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole‐type inhibitors are energetically poised to report faithfully on mannosidase transition‐state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including β‐mannanases from families GH26 and GH113. Isofagomine‐type inhibitors are poor mimics of transition‐state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar‐shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active‐site residues involved in substrate recognition.     

Evidence for a Boat Conformation at the Transition State of GH76 α‐1,6‐Mannanases—Key Enzymes in Bacterial and Fungal Mannoprotein Metabolism

α‐Mannosidases and α‐mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α‐mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α‐mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an ^OS₂ conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B_2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B_2,5 conformation the most energetically stable on‐enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through ^OS₂ and B_2,5^≠ conformations, information that should inspire the development of new antifungal agents.