N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7–2.1 Å reveal a ( β / α ) 8 barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc 1/3 Man 9 GlcNAc 2 yields Glc 1/3 -Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

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.

Hydrolysis of β-d-mannosides by β-mannosidases typically proceeds via a B 2,5 transition state conformation for the pyranoside ring, while that of β-d-glucosides by β-glucosidases proceeds through a distinct 4 H 3 transition state conformation. However, rice Os7BGlu26 β-glycosidase hydrolyzes 4-nitrophenyl β-d-glucoside and β-d-mannoside with similar efficiencies. The origin of this dual substrate specificity was investigated by kinetic, structural, and computational approaches. The glycosidase inhibitors glucoimidazole and mannoimidazole inhibited Os7BGlu26 with K i values of 2.7 nM and 10.4 μM, respectively. In X-ray crystal structures of complexes with Os7BGlu26, glucoimidazole bound to the active site in a 4 E conformation, while mannoimidazole bound in a B 2,5 conformation, suggesting different transition state conformations. Moreover, calculation of quantum mechanics/molecular mechanics (QM/MM) free energy landscapes showed that 4-nitrophenyl β-d-glucoside adopts a 1 S 3/4 E conformation in the Michaelis complex, while 4-nitrophenyl β-d-mannoside adopts a 1 S 5/B 2,5 conformation. The QM/MM simulations of Os7BGlu26 catalysis of hydrolysis also supported the itineraries of 1 S 3 → 4 E/4 H 3 ⧧ → 4 C 1 for β-d-glucosides and 1 S 5 → B 2,5 ⧧ → O S 2 for β-d-mannosides, thereby revealing that a single glycoside hydrolase can hydrolyze glycosides of different configurations via distinct transition state pyranoside conformations.

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