Abstract
Colonic bacteria, exemplified by Bacteroides thetaiotaomicron, play a key role in maintaining human health by harnessing large families of glycoside hydrolases (GHs) to exploit dietary polysaccharides and host glycans as nutrients. Such GH family expansion is exemplified by the 23 family GH92 glycosidases encoded by the B. thetaiotaomicron genome. Here we show that these are α-mannosidases that act via a single displacement mechanism to utilize host N-glycans. The three-dimensional structure of two GH92 mannosidases defines a family of two-domain proteins in which the catalytic center is located at the domain interface, providing acid (glutamate) and base (aspartate) assistance to hydrolysis in a Ca2+-dependent manner. The three-dimensional structures of the GH92s in complex with inhibitors provide insight into the specificity, mechanism and conformational itinerary of catalysis. Ca2+ plays a key catalytic role in helping distort the mannoside away from its ground-state 4C1 chair conformation toward the transition state.
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Acknowledgements
We thank S. Withers (University of British Columbia) for compound 9. The Biotechnology and Biological Sciences Research Council of the UK is thanked for funding. M.D.L.S. is a European Molecular Biology Laboratory long-term fellowship holder, and G.J.D. is a Royal Society/Wolfson Research Merit Award recipient. C.D. was supported by a Marie Curie European Reintegration grant within the 7th European Community Framework Programme. Y.Z. is supported by the Department for Innovation, Universities and Skills (DIUS), UK, Newcastle University and the Chinese Scholarship Council. S.J.W. thanks the Australian Research Council for funding support. K.W.M. acknowledges the support of US National Institutes of Health grants GM047533, RR005351 and DK075322. S.C. is supported by a postdoctoral fellowship from the Agence Nationale de Recherche sur le Sida et les Hépatities Virales (ANRS). A.S. is supported by Centre National de la Recherche Scientifique.
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H.J.G. and G.J.D. designed the experiments. Y.Z., M.D.L.S., A.J.T., C.D., N.S., Y.X. and K.W.M. performed or supervised molecular biology, kinetics, glycan analysis and X-ray crystallography. S.C., A.S., Z.D. and S.J.W. synthesized ligands. H.J.G., G.J.D. and S.J.W., aided by the other authors, wrote the paper.
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Zhu, Y., Suits, M., Thompson, A. et al. Mechanistic insights into a Ca2+-dependent family of α-mannosidases in a human gut symbiont. Nat Chem Biol 6, 125–132 (2010). https://doi.org/10.1038/nchembio.278
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DOI: https://doi.org/10.1038/nchembio.278
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