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

alpha-Mannosidases and alpha-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of alpha-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 alpha-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an S-O(2) conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B-2,5(not equal) 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,B-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(not equal) conformations, information that should inspire the development of new antifungal agents.