Mechanistic insights into a Ca2+-dependent family of α- mannosidases in a human gut symbiont

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 C1 chair conformation toward the transition state. © 2010 Nature America, Inc. All rights reserved. *Correspondence and requests for materials should be addressed to H.J.G. or G.J.D. davies@ysbl.york. ac.uk or hgilbert@ccrc.uga. edu. 6Present address: INRA, UMR792 Ingénierie des Systèmes biologiques et des Procédés, F-31400 Toulouse; Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse; CNRS, UMR5504, F-31400 Toulouse, France. 7These authors contributed equally to this work. Accession codes. Protein Data Bank: The crystal structures of Bt3990 by itself, and with compounds 6, 7, 5, 4, and 4 with calcium, have been deposited under accession codes 2WVX, 2WZS, 2WVZ, 2WW0, 2WW1 and 2WW3, respectively. The crystal structures of Bt2199 by itself and with compound 5 have been deposited under accession codes 2WVY and 2WW2, respectively. Author contributions 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. Additional information Supplementary information and chemical compound information is available online at http://www.nature. com/ naturechemicalbiology/. Reprints and permissions information is available online at http://npg.nature. com/reprintsandpermissions/. NIH Public Access Author Manuscript Nat Chem Biol. Author manuscript; available in PMC 2014 March 04. Published in final edited form as: Nat Chem Biol. 2010 February ; 6(2): 125–132. doi:10.1038/nchembio.278. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript The human large bowel microbiota, consisting of ~1014 bacteria, contributes to human nutrition and health1. It is estimated that ~10% of caloric intake is derived from plant polysaccharides that are deconstructed exclusively by enzymes produced by the microbiota2. The large bowel microbiota has a generic influence on host-nutrient partitioning, particularly with respect to carbohydrates and fats. An example of this modulation is provided by the observation that the transfer of this bacterial community from a genetically obese mouse to a normal animal induces obesity in its new host3. The colonic microbiota can also influence the host immune system, enhancing the health of the host4,5. Furthermore, butyrate, an end product of colonic fermentation, is thought to have antineoplastic properties and thus plays a role in maintaining a healthy gut6. Reflecting the critical role the large bowel microbiota plays in maintaining human health, there have been numerous attempts to manipulate the composition of this ecosystem through dietary and probiotic intervention6. Such strategies have been hampered by a paucity of knowledge of the biochemistry of these micro-organisms; particularly with respect to their utilization of complex carbohydrates as nutrients. Researchers have started to unravel the genetic basis for the function of the microbiota. Thus, the genome sequences of selected dominant colonic bacteria are available7–9, and metagenomics is starting to yield more extensive information on the complete microbiota10,11. Commensurate with their nutritional flexibility, the genomes of colonic bacteria, exemplified by B. thetaiotaomicron, have revealed an array of genes encoding glycoside hydrolases (>200). The vast majority of these enzymes are in glycoside hydrolase families (GHs) that contain mainly exo-acting enzymes, which may suggest that the organism is able to use the saccharide decorations appended to the backbone of structural polysaccharides and glycoproteins8. This expansion in exo-GHs is particularly evident in family GH92, which contains 23 members; however, only α-1,2mannosidase activity has been ascribed to this family12. There are few α-linked mannosyl linkages in nature (Fig. 1), and it is unclear whether the evolutionary drivers for the observed expansion of GH92 in B. thetaiotaomicron reflect substrate or regulatory complexity. Here we present the analysis of 22 of the 23 GH92 enzymes encoded by the genome of B. thetaiotaomicron. We show that these enzymes are Ca2+-dependent α-mannosidases that perform catalysis with inversion of anomeric configuration. The three-dimensional structures of GH92 α-1,2-mannosidases Bt3990 and Bt2199 reveal bimodular proteins in which an N-terminal β-sheet domain is appended to an (α/α)6 barrel catalytic module in which a shallow surface depression between the two domains comprises the active site. The three-dimensional analysis of inhibitor complexes provides insight into the likely conformational itinerary for mannoside hydrolysis. This study, in addition to providing a biochemical basis for expansion of the GH92 family in a dominant colonic bacterium, informs the use of small-molecule α-mannosidase inhibitors in probing the functional significance of this enzyme family in the survival of B. thetaiotaomicron in the human colon.