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)
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
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
Data acquisition
Detectors
Diagnostics
Health Physics
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Immacolata
Venditto
,
Ana S.
Luis
,
Maja
Rydahl
,
Julia
Schückel
,
Vânia O.
Fernandes
,
Silvia
Vidal-Melgosa
,
Pedro
Bule
,
Arun
Goyal
,
Virginia M. R.
Pires
,
Catarina G.
Dourado
,
Luís M. A.
Ferreira
,
Pedro M.
Coutinho
,
Bernard
Henrissat
,
J. Paul
Knox
,
Arnaud
Baslé
,
Shabir
Najmudin
,
Harry J.
Gilbert
,
William G. T.
Willats
,
Carlos M. G. A.
Fontes
Diamond Proposal Number(s):
[9948]
Abstract: The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBMfamilies that targeted beta-glucans, beta-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize beta-glucans and beta-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.
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Jun 2016
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I03-Macromolecular Crystallography
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Abstract: A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nanomachines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such as Ruminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. Over 220 genes encoding dockerin-containing proteins have been identified in the R. flavefaciens genome. The dockerin-containing enzymes can be incorporated into the primary scaffoldin (ScaA), which in turn can bind to adaptor scaffoldins (ScaB or ScaC) and subsequently to anchoring scaffoldin (ScaE), thereby attaching the whole complex to the cell surface. However, unlike other cellulosomes such as that from Clostridium thermocellum, the Ruminococcus species lack a specific carbohydrate-binding module (CBM) on ScaA which recruits the entire complex onto the surface of the substrate. Instead, a cellulose-binding protein, CttA, comprising two putative tandem novel carbohydrate-binding modules and a C-terminal X-dockerin module, which can bind to the cohesin of ScaE, may mediate the attachment of bacterial cells to cellulose. Here, the expression, purification and crystallization of the carbohydrate-binding modular part of the CttA from R. flavefaciens are described. X-ray data have been collected to resolutions of 3.23 and to 1.61 Å in space groups P3121 or P3221 and P21, respectively. The structure was phased using bound iodide from the crystallization buffer by SAD experiments.
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Jun 2015
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I02-Macromolecular Crystallography
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Abstract: Anaerobic bacteria organize carbohydrate-active enzymes into a multi-component complex, the cellulosome, which degrades cellulose and hemicellulose highly efficiently. Genome sequencing of Ruminococcus flavefaciens FD-1 offers extensive information on the range and diversity of the enzymatic and structural components of the cellulosome. The R. flavefaciens FD-1 genome encodes over 200 dockerin-containing proteins, most of which are of unknown function. One of these modular proteins comprises a glycoside hydrolase family 5 catalytic module (GH5) linked to an unclassified carbohydrate-binding module (CBM-Rf1) and a dockerin. The novel CBM-Rf1 has been purified and crystallized. The crystals belonged to the trigonal space group R32:H. The CBM-Rf1 structure was determined by a multiple-wavelength anomalous dispersion experiment using AutoSol from the PHENIX suite using both selenomethionyl-derivative and native data to resolutions of 2.28 and 2.0 Å, respectively.
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Dec 2014
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: Cellulases catalyze the hydrolysis of cellulose, the major constituent of plant biomass and the most abundant organic polymer on earth. Cellulases are modular enzymes containing catalytic domains connected, via linker sequences, to noncatalytic carbohydrate-binding modules (CBMs). A putative modular endo-[beta]-1,4-glucanase (BhCel5B) is encoded at locus BH0603 in the genome of Bacillus halodurans. It is composed of an N-terminal glycoside hydrolase family 5 catalytic module (GH5) followed by an immunoglobulin-like module and a C-terminal family 46 CBM (BhCBM46). Here, the crystallization and preliminary X-ray diffraction analysis of the trimodular BhCel5B are reported. The crystals of BhCel5B belonged to the orthorhombic space group P2121 2 and data were processed to a resolution of 1.64 Å. A molecular-replacement solution has been found.
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Dec 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8425]
Abstract: Plant cell-wall polysaccharides offer an abundant energy source utilized by many microorganisms, thus playing a central role in carbon recycling. Aerobic microorganisms secrete carbohydrate-active enzymes (CAZymes) that catabolize this composite structure, comprising cellulose, hemicellulose and lignin, into simple compounds such as glucose. Carbohydrate-binding modules (CBMs) enhance the efficacy of associated CAZYmes. They are organized into families based on primary-sequence homology. CBM family 46 contains more than 40 different members, but has yet to be fully characterized. Here, a recombinant derivative of the C-terminal family 46 CBM module (BhCBM46) of Bacillus halodurans endo-[beta]-1,4-glucanase B (CelB) was overexpressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography. Preliminary structural characterization was carried out on BhCBM46 crystallized in different conditions. The crystals of BhCBM46 belonged to the tetragonal space group I4122. Data were collected for the native form and a selenomethionine derivative to 2.46 and 2.3 Å resolution, respectively. The BhCBM46 structure was determined by a single-wavelength anomalous dispersion experiment using AutoSol from the PHENIX suite.
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Jun 2014
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Ana S.
Luis
,
Immacolata
Venditto
,
Max J.
Temple
,
Artur
Rogowski
,
Arnaud
Basle
,
Jie
Xue
,
J. Paul
Knox
,
Jose A. M.
Prates
,
Luis M. A.
Ferreira
,
Carlos M. G. A.
Fontes
,
Shabir
Najmudin
,
Harry J.
Gilbert
Diamond Proposal Number(s):
[1221]
Open Access
Abstract: Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with β1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.
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Feb 2013
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