B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Mélanie
Loiodice
,
Elodie
Drula
,
Zak
Mciver
,
Svetlana
Antonyuk
,
Arnaud
Basle
,
Marcelo
Lima
,
Edwin A.
Yates
,
Dominic P.
Byrne
,
Jamie
Coughlan
,
Andrew
Leech
,
Shahram
Mesdaghi
,
Daniel J.
Rigden
,
Sophie
Drouillard
,
William
Helbert
,
Bernard
Henrissat
,
Nicolas
Terrapon
,
Gareth S. A.
Wright
,
Marie
Couturier
,
Alan
Cartmell
Diamond Proposal Number(s):
[18598, 30305, 21970, 32677, 28406]
Open Access
Abstract: Acidic glycans are essential for the biology of multicellular eukaryotes. To utilize them, microbial life including symbionts and pathogens has evolved polysaccharide lyases (PL) that cleave their 1,4 glycosidic linkages via a β-elimination mechanism. PL family 33 (PL33) enzymes have the unusual ability to target a diverse range of glycosaminoglycans (GAGs), as well as the bacterial polymer, gellan gum. In order to gain more detailed insight into PL33 activities we recombinantly expressed 10 PL33 members derived from all major environments and further elucidated the detailed biochemical and biophysical properties of five, showing that their substrate specificity is conferred by variations in tunnel length and topography. The key amino acids involved in catalysis and substrate interactions were identified, and employing a combination of complementary biochemical, structural, and modeling approaches, we show that the tunnel topography is induced by substrate binding to the glycan. Structural and bioinformatic analyses revealed that these features are conserved across several lyase families as well as in mammalian GAG epimerases.
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Feb 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19951]
Open Access
Abstract: Background: Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes. Results: We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. Conclusions: The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.
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May 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the ‘sulfation problem’ is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.
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Jan 2020
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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
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948, 13587]
Open Access
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.
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Jun 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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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.
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Apr 2017
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[1960, 7854, 9948]
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.
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Mar 2017
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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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