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Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	A surface endogalactanase in Bacteroides thetaiotaomicron confers keystone status for arabinogalactan degradation Alan Cartmell , Jose Muñoz-Muñoz , Jonathon A. Briggs , Didier A. Ndeh , Elisabeth C. Lowe , Arnaud Basle , Nicolas Terrapon , Katherine Stott , Tiaan Heunis , Joe Gray , Li Yu , Paul Dupree , Pearl Z. Fernandes , Sayali Shah , Spencer J. Williams , Aurore Labourel , Matthias Trost , Bernard Henrissat , Harry J. Gilbert Nature Microbiology 3, 1314 - 1326 DOI: 10.1038/s41564-018-0258-8 Diamond Proposal Number(s): [1960, 7854, 9948] Show Abstract ▼ Abstract: Glycans are major nutrients for the human gut microbiota (HGM). Arabinogalactan proteins (AGPs) comprise a heterogenous group of plant glycans in which a β1,3-galactan backbone and β1,6-galactan side chains are conserved. Diversity is provided by the variable nature of the sugars that decorate the galactans. The mechanisms by which nutritionally relevant AGPs are degraded in the HGM are poorly understood. Here we explore how the HGM organism Bacteroides thetaiotaomicron metabolizes AGPs. We propose a sequential degradative model in which exo-acting glycoside hydrolase (GH) family 43 β1,3-galactanases release the side chains. These oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or GH. We identified two GHs that establish two previously undiscovered GH families. The crystal structures of the exo-β1,3-galactanases identified a key specificity determinant and departure from the canonical catalytic apparatus of GH43 enzymes. Growth studies of Bacteroidetes spp. on complex AGP revealed 3 keystone organisms that facilitated utilization of the glycan by 17 recipient bacteria, which included B. thetaiotaomicron. A surface endo-β1,3-galactanase, when engineered into B. thetaiotaomicron, enabled the bacterium to utilize complex AGPs and act as a keystone organism.	Nov 2018
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides Ana S. Luis , Jonathon Briggs , Xiaoyang Zhang , Benjamin Farnell , Didier Ndeh , Aurore Labourel , Arnaud Basle , Alan Cartmell , Nicolas Terrapon , Katherine Stott , Elisabeth C. Lowe , Richard Mclean , Kaitlyn Shearer , Julia Schückel , Immacolata Venditto , Marie-Christine Ralet , Bernard Henrissat , Eric C. Martens , Steven C. Mosimann , D. Wade Abbott , Harry J. Gilbert Nature Microbiology 344 DOI: 10.1038/s41564-017-0079-1 Diamond Proposal Number(s): [1960, 7854, 9948] Show Abstract ▼ Abstract: The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilization loci (PULs). In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by different pectin domains have been identified; however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I is particularly complex. This system contains several glycoside hydrolases that trim the remnants of other pectin domains attached to rhamnogalacturonan-I, and nine enzymes that contribute to the degradation of the backbone that makes up a rhamnose-GalA repeating unit. The catalytic properties of the pectin-degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms.	Dec 2017
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans Alan Cartmell , Elisabeth C. Lowe , Arnaud Basle , Susan J. Firbank , Didier A. Ndeh , Heath Murray , Nicolas Terrapon , Vincent Lombard , Bernard Henrissat , Jeremy E. Turnbull , Mirjam Czjzek , Harry J. Gilbert , David N. Bolam Proceedings Of The National Academy Of Sciences 114, 7037 - 7042 DOI: 10.1073/pnas.1704367114 Diamond Proposal Number(s): [311, 9948] Open Access Show Abstract ▼ Abstract: The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community.	Jul 2017
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins Jose Munoz-Munoz , Alan Cartmell , Nicolas Terrapon , Arnaud Basle , Bernard Henrissat , Harry J. Gilbert Journal Of Biological Chemistry DOI: 10.1074/jbc.M117.794578 Diamond Proposal Number(s): [9948, 13587] Open Access Show Abstract ▼ Abstract: The human gut microbiota utilizes complex carbohydrates as major nutrients. The requirement for efficient glycan degrading systems exerts a major selective selection pressure on this microbial community. Thus, we propose that this microbial ecosystem represents a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis we screened the potential enzymatic functions of hypothetical proteins encoded by genes of Bacteroides thetaiotaomicron that were upregulated by arabinogalactan arabinogalactan proteins or AGPs. Although AGPs are ubiquitous in plants, there is a paucity of information on their detailed structure, the function of these glycans in planta and the mechanisms by which they are depolymerized in microbial ecosystems. Here we have discovered a new polysaccharide lyase family that is specific for the L-rhamnose-alpha1,4-D-glucuronic acid linkage that caps the side chains of complex AGPs. The reaction product generated by the lyase, delta4,5-unsaturated uronic acid, is removed from AGP by a glycoside hydrolase located in family GH105, producing the final product 4-deoxy-β-L-threo-hex-4-enepyranosyl-uronic acid. The crystal structure of a member of the novel lyase family revealed a catalytic domain that displays an (alpha/alpha6)6 barrel fold. In the centre of the barrel is a deep pocket, which, based on mutagenesis data and amino acid conservation, comprises the active site of the lyase. A tyrosine is the proposed catalytic base in the beta-elimination reaction. This study illustrates how highly complex glycans can be used as a scaffold to discover new enzyme families within microbial ecosystems where carbohydrate metabolism is a major evolutionary driver.	Jun 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	Unusual active site location and catalytic apparatus in a glycoside hydrolase family Jose Munoz-Munoz , Alan Cartmell , Nicolas Terrapon , Bernard Henrissat , Harry J. Gilbert Proceedings Of The National Academy Of Sciences 447 DOI: 10.1073/pnas.1701130114 Show Abstract ▼ Abstract: The human gut microbiota use complex carbohydrates as major nutrients. The requirement for an efficient glycan degrading systems exerts a major selection pressure on this microbial community. Thus, we propose that these bacteria represent a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis, we focused on enzymes that hydrolyze rhamnosidic bonds, as cleavage of these linkages is chemically challenging and there is a paucity of information on l-rhamnosidases. Here we screened the activity of enzymes derived from the human gut microbiota bacterium Bacteroides thetaiotaomicron, which are up-regulated in response to rhamnose-containing glycans. We identified an α-l-rhamnosidase, BT3686, which is the founding member of a glycoside hydrolase (GH) family, GH145. In contrast to other rhamnosidases, BT3686 cleaved l-Rha-α1,4–d-GlcA linkages through a retaining double-displacement mechanism. The crystal structure of BT3686 showed that the enzyme displayed a type A seven-bladed β-propeller fold. Mutagenesis and crystallographic studies, including the structure of BT3686 in complex with the reaction product GlcA, revealed a location for the active site among β-propeller enzymes cited on the posterior surface of the rhamnosidase. In contrast to the vast majority of GH, the catalytic apparatus of BT3686 does not comprise a pair of carboxylic acid residues but, uniquely, a single histidine functions as the only discernable catalytic amino acid. Intriguingly, the histidine, His48, is not invariant in GH145; however, when engineered into structural homologs lacking the imidazole residue, α-l-rhamnosidase activity was established. The potential contribution of His48 to the catalytic activity of BT3686 is discussed.	Apr 2017
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Complex pectin metabolism by gut bacteria reveals novel catalytic functions Didier Ndeh , Artur Rogowski , Alan Cartmell , Ana S. Luis , Arnaud Basle , Joseph Gray , Immacolata Venditto , Jonathon Briggs , Xiaoyang Zhang , Aurore Labourel , Nicolas Terrapon , Fanny Buffetto , Sergey Nepogodiev , Yao Xiao , Robert A. Field , Yanping Zhu , Malcolm A. O’neill , Breeanna R. Urbanowicz , William S. York , Gideon J. Davies , D. Wade Abbott , Marie-Christine Ralet , Eric C. Martens , Bernard Henrissat , Harry J. Gilbert Nature 411 DOI: 10.1038/nature21725 Diamond Proposal Number(s): [1960, 7854, 9948] Show Abstract ▼ Abstract: The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.	Mar 2017
I02-Macromolecular Crystallography	Glycan complexity dictates microbial resource allocation in the large intestine Artur Rogowski , Jonathon A. Briggs , Jennifer C. Mortimer , Theodora Tryfona , Nicolas Terrapon , Elisabeth C. Lowe , Arnaud Baslé , Carl Morland , Alison M. Day , Hongjun Zheng , Theresa E. Rogers , Paul Thompson , Alastair R. Hawkins , Madhav P. Yadav , Bernard Henrissat , Eric C. Martens , Paul Dupree , Harry J. Gilbert , David N. Bolam Nature Communications 6, 7481 DOI: 10.1038/ncomms8481 PMID: 26112186 Open Access Show Abstract ▼ Abstract: The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.	Jun 2015

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