Conformational Analysis DOI: 10.1002/ange.201410502 Evidence for a Boat Conformation at the Transition State of GH76 a- 1,6-Mannanases—Key Enzymes in Bacterial and Fungal Mannoprotein Metabolism** AndrewJ. Thompson, Gaetano Speciale, Javier Iglesias-Fernµndez, Zalihe Hakki, Tyson Belz, Alan Cartmell, Richard J. Spears, Emily Chandler, Max J. Temple, Judith Stepper, Harry J. Gilbert, Carme Rovira,* Spencer J. Williams,* and GideonJ. Davies* Abstract: a-Mannosidases and a-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of a-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 a-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an O S 2 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 O S 2 and B 2,5 ° conformations, information that should inspire the development of new antifungal agents. Enzymes catalyzing the hydrolysis of glycosidic bonds within a-mannoside-based glycans and glycoconjugates (a-manno- sidases and a-mannanases) are of great interest due to their roles in glycoprotein maturation, [1] cell wall assembly in fungi, [2] and processing of complex dietary polysaccharides by the human gut microbiota. [3] Relatively little attention has been directed at endo-acting a-mannosidases and a-manna- nases, enzymes that play major roles in eukaryotic N-glycan processing [4] and in the metabolism and deconstruction of fungal cell wall a-mannans. [3] Endo-a-mannanases are grouped into two families within the CAZy [5] sequence- based classification (http://www.cazy.org): GH99, which includes both mammalian and bacterial endo-a-mannosidases and endo-a-mannanases; and GH76, featuring endo-acting bacterial a-1,6-mannanases [3, 5] and fungal transglycosi- dases. [2, 6] A growing body of literature has reported detailed characterization of various GH99 endo-a-mannosidases in terms of function, [4a] cellular localization, [7] structure, [8] and the development of effective inhibitors that are active within cells; [9] by comparison our knowledge and understanding of the biological role of GH76 enzymes has trailed. While several GH76 structures are available, less is known of the biochemistry and mechanism of these enzymes, no inhibitors have been reported and nothing is known of the conforma- tional changes occurring during catalysis. Most a- and b-mannosidases perform catalysis through one of two conformational itineraries. X-ray structures of insightful ligand complexes, dovetailed with computational analyses of conformational free-energy landscapes (FEL) of inhibitors on- or off-enzyme, have allowed assignment of an O S 2 $B 2,5 ° $ 1 S 5 itinerary to a- and b-mannosidases of families GH2, 26, 38, 92, and 113. [10] Alternatively, an unusual “southern hemisphere” itinerary, 3 S 1 ! 3 H 4 ° ! 1 C 4 , has been assigned to exo-acting a-mannosidases of family GH47. [11] Much effort has gone into the synthesis of enzyme inhibitors that either report on, or reflect, these conformational path- ways. We recently provided evidence that the mannosidase inhibitor, mannoimidazole, is an exquisitely informative conformational probe, owing to the relatively small energy differences and barrier-less interconversion between ground- state 4 H 3 or 3 H 4 conformations, and higher energy 2,5 B or B 2,5 conformations. [10e] In contrast, the azasugar isofagomine [*] Dr. A.J. Thompson, R. J. Spears, E. Chandler, Dr. J. Stepper, Prof. G. J. Davies Department of Chemistry, University of York Heslington, York, YO10 5DD (UK) E-mail: gideon.davies@york.ac.uk G. Speciale, Z. Hakki, T. Belz, Dr. A. Cartmell, Prof. S.J. Williams School of Chemistry and Bio21 Molecular Science and Biotech- nology Institute, University of Melbourne Parkville, Vic 3010 (Australia) E-mail: sjwill@unimelb.edu.au J. Iglesias-Fernµndez, Prof. C. Rovira Departament de Química Orgànica and Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona 08028 Barcelona (Spain) E-mail: c.rovira@ub.edu M. J. Temple, Prof. H. J. Gilbert Institute for Cell and Molecular Biosciences The Medical School Newcastle University, Newcastle upon Tyne, NE2 4HH (UK) [**] We thank the Australian Research Council, the UK Biotechnology and Biological Sciences Research Council, Amicus Therapeutics, the Spanish Ministry of Economy and Competitiveness, the Generalitat de Catalunya, and the European Research Council. We also acknowledge the staff of the Diamond Light Source (Didcot (UK)) for provision of beamline facilities, and the support, technical expertise, and assistance provided by the Barcelona Supercomput- ing Center: Centro Nacional de Supercomputación. Supporting information for this article is given via a link at the end of the document. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201410502. A ngewandte Chemi e 1 Angew. Chem. 2015, 127,1–6  2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü