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Glycoside hydrolase family 99 (GH99) was created to categorize sequence-related glycosidases possessing endo-α-mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N-glycan precursors (Glc1-3Man9GlcNAc2), releasing mono-, di- and triglucosylated-mannose (Glc1-3-1,3-Man). GH99 family members have recently been implicated in the ability of Bacteroides spp., present within the gut microbiota, to metabolize fungal cell wall α-mannans, releasing α-1,3-mannobiose by hydrolysing αMan-1,3-αMan→1,2-αMan-1,2-αMan sequences within branches off the main α-1,6-mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel endo-α-1,2-mannanase activity of bacterial GH99 enzymes from Bacteroides thetaiotaomicron and xylanisolvens. These data reveal an approximate 5 kJ mol−1 preference for mannose-configured substrates in the −2 subsite (relative to glucose), which inspired the development of a new inhibitor, α-mannopyranosyl-1,3-isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X-ray structures of ManIFG or a substrate in complex with wild-type or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to D-mannose- rather than D-glucose-configured substrates. [ABSTRACT FROM AUTHOR]

A correction to the article "Human Gut Bacteroidetes Can Utilize Yeast Mannan Through A Selfish Mechanism" by Fiona Cuskin, Max J. Temple and Yanping Zhu published in "Nature" Vol 517 2015 issue is presented.

N-Glycans direct protein function, stability, folding and targeting, and influence immunogenicity. While most glycosidases that process N-glycans cleave a single sugar residue at a time, enzymes from glycoside hydrolase family 99 are endo-acting enzymes that cleave within complex N-glycans. Eukaryotic Golgi endo-1,2-α-mannosidase cleaves glucose-substituted mannose within immature glucosylated high-mannose N-glycans in the secretory pathway. Certain bacteria within the human gut microbiota produce endo-1,2-α-mannanase, which cleaves related structures within fungal mannan, as part of nutrient acquisition. An unconventional mechanism of catalysis was proposed for enzymes of this family, hinted at by crystal structures of imino/azasugars complexed within the active site. Based on this mechanism, we developed the synthesis of two glycosides bearing a spiro-epoxide at C-2 as electrophilic trap, to covalently bind a mechanistically important, conserved GH99 catalytic residue. The spiro-epoxyglycosides are equipped with a fluorescent tag, and following incubation with recombinant enzyme, allow concentration, time and pH dependent visualization of the bound enzyme using gel electrophoresis., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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 (O) S2 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 (O) S2 and B2,5 (≠) conformations, information that should inspire the development of new antifungal agents., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)