I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Justina
Briliūtė
,
Paulina A.
Urbanowicz
,
Ana S.
Luis
,
Arnaud
Basle
,
Neil
Paterson
,
Osmond
Rebello
,
Jenifer
Hendel
,
Didier A.
Ndeh
,
Elisabeth C.
Lowe
,
Eric C.
Martens
,
Daniel I. R.
Spencer
,
David N.
Bolam
,
Lucy I.
Crouch
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.
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Jun 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[1960, 7854, 9948]
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.
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Nov 2018
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[1960, 7854, 9948]
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.
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Dec 2017
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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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
Diamond Proposal Number(s):
[311, 9948]
Open Access
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.
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Jul 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948, 13587]
Abstract: Glycans are major nutrients available to the human gut microbiota (HGM). The Bacteroides are generalist glycan degraders and this function is mediated largely by polysaccharide utilization loci (PULs). The genomes of several Bacteroides species contain a PUL, PUL1,6-beta;-glucan, that was predicted to target mixed linked plant 1,3;1,4-beta-glucans. To test this hypothesis we characterized the proteins encoded by this locus in Bacteroides thetaiotaomicron, a member of the HGM. We show here that PUL1,6-β-glucan does not orchestrate the degradation of a plant polysaccharide but targets a fungal cell wall glycan, 1,6-beta-glucan, which is a growth substrate for the bacterium. The locus is upregulated by 1,6-beta-glucan, and encodes two enzymes, a surface endo-1,6-beta-glucanase, BT3312, and a periplasmic beta-glucosidase that targets primarily 1,6-beta-glucans. The non-catalytic proteins encoded by PUL1,6-beta-glucan target 1,6-beta-glucans and comprise a surface glycan binding protein and a SusD homologue that delivers glycans to the outer membrane transporter. We identified the central role of the endo-1,6-beta-glucanase in 1,6-beta-glucan depolymerization by deleting bt3312, which prevented the growth of B. thetaiotaomicron on 1,6-beta-glucan. The crystal structure of BT3312 in complex with β-glucosyl-1,6-deoxynojirimycin, revealed a TIM barrel catalytic domain that contains a deep substrate binding cleft tailored to accommodate the hook-like structure adopted by 1,6-beta-glucan. Specificity is driven by the complementarity of the enzyme active site cleft and the conformation of the substrate. We also noted that PUL1,6-beta-glucan is syntenic to many PULs from other Bacteroidetes suggesting that utilization of yeast and fungal cell wall 1,6-beta-glucans is a widespread adaptation within the human microbiota.
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May 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. beta-mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although a family of glycoside phosphorylases, GH130, have also been shown to target beta-1,2 and beta-1,4 mannosidic linkages. In these phosphorylases bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA03624, which lack the phosphate binding residues are indeed betamannosidases that hydrolyse beta-1,2-mannosidic linkages through an inverting mechanism. As the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerisation of yeast alpha-mannans, it is likely that the two enzymes target the beta-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the -1 and +1 subsites showed a pair of glutamates, Glu227 and Glu268 hydrogen bond to O1 of alpha-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and beta-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys199 in BT3780, as a key specificity determinant for beta-1,2-mannosidic linkages.
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Aug 2015
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I02-Macromolecular Crystallography
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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
Open Access
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.
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Jun 2015
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I03-Macromolecular Crystallography
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Fiona
Cuskin
,
Elisabeth C.
Lowe
,
Max J.
Temple
,
Yanping
Zhu
,
Elizabeth A.
Cameron
,
Nicholas A.
Pudlo
,
Nathan T.
Porter
,
Karthik
Urs
,
Andrew J.
Thompson
,
Alan
Cartmell
,
Artur
Rogowski
,
Brian S.
Hamilton
,
Rui
Chen
,
Thomas J.
Tolbert
,
Kathleen
Piens
,
Debby
Bracke
,
Wouter
Vervecken
,
Zalihe
Hakki
,
Gaetano
Speciale
,
Jose L.
Munoz-Munoz
Diamond Proposal Number(s):
[7864]
Abstract: Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a selfish model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.
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Jan 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Rohan J.
Williams
,
Javier
Iglesias-Fernández
,
Judith
Stepper
,
Adam
Jackson
,
Andrew
Thompson
,
Elisabeth C.
Lowe
,
John
White
,
Harry J.
Gilbert
,
Carme
Rovira
,
Gideon J.
Davies
,
Spencer J.
Williams
Diamond Proposal Number(s):
[7864]
Open Access
Abstract: 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.
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Jan 2014
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1221]
Abstract: Signaling across the membrane in response to extracellular stimuli is essential for survival of all cells. In bacteria, responses to environmental changes are predominantly mediated by two-component systems, which are typically composed of a membrane-spanning sensor histidine kinase and a cytoplasmic response regulator. In the human gut symbiont Bacteroides thetaiotaomicron, hybrid two-component systems are a key part of the bacterium’s ability to sense and degrade complex carbohydrates in the gut. Here, we identify the activating ligand of the hybrid two-component system, BT4663, which controls heparin and heparan sulfate acquisition and degradation in this prominent gut microbe, and report the crystal structure of the extracellular sensor domain in both apo and ligand-bound forms. Current models for signal transduction across the membrane involve either a piston-like or rotational displacement of the transmembrane helices to modulate activity of the linked cytoplasmic kinases. The structures of the BT4663 sensor domain reveal a significant conformational change in the homodimer on ligand binding, which results in a scissor-like closing of the C-termini of each protomer. We propose this movement activates the attached intracellular kinase domains and represents an allosteric mechanism for bacterial transmembrane signaling distinct from previously described models, thus expanding our understanding of signal transduction across the membrane, a fundamental requirement in many important biological processes.
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May 2012
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