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

• Published in: Angewandte Chemie International Edition 2015-04, Vol.54 (18), p.5378-5382
• Main Authors: Thompson, Andrew J., Speciale, Gaetano, Iglesias-Fernández, Javier, Hakki, Zalihe, Belz, Tyson, Cartmell, Alan, Spears, Richard J., Chandler, Emily, Temple, Max J., Stepper, Judith, Gilbert, Harry J., Rovira, Carme, Williams, Spencer J., Davies, Gideon J.
• Format: Article
• Language: English
• Subjects: Aza Compounds - chemical synthesis; Aza Compounds - chemistry; Aza Compounds - pharmacology; Bacillus - enzymology; Bacteria; Bacterial Proteins - metabolism; Boats; Candida albicans - enzymology; carbohydrates; Cellular; computational chemistry; conformational analysis; Distortion; enzymatic mechanisms; Enzyme Inhibitors - chemical synthesis; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Enzymes; Fungal Proteins - metabolism; Fungi; glycosidase inhibitors; Imino Sugars - chemical synthesis; Imino Sugars - chemistry; Imino Sugars - pharmacology; Inhibitors; Mannosidases - antagonists & inhibitors; Mannosidases - chemistry; Models, Molecular; Protein Conformation; Rendering; Walls
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201410502

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 B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on‐enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through OS2 and B2,5≠ conformations, information that should inspire the development of new antifungal agents. Family GH76­ endo‐α‐mannanases participate in construction and breakdown of fungal cell wall mannoprotein. A combined synthetic, structural, and theoretical study discloses the first inhibitors of this family of enzymes and quantifies how the enzyme distorts an azasugar inhibitor into a transition‐state‐mimicking boat conformation.