I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
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.
|
Nov 2018
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: The biological conversion of lignocellulosic matter into high-value chemicals or biofuels is of increasing industrial importance as the sector slowly transitions away from nonrenewable sources. Many industrial processes involve the use of cellulolytic enzyme cocktails – a selection of glycoside hydrolases and, increasingly, polysaccharide oxygenases – to break down recalcitrant plant polysaccharides. ORFs from the genome of Teredinibacter turnerae, a symbiont hosted within the gills of marine shipworms, were identified in order to search for enzymes with desirable traits. Here, a putative T. turnerae glycoside hydrolase from family 8, hereafter referred to as TtGH8, is analysed. The enzyme is shown to be active against β-1,4-xylan and mixed-linkage (β-1,3,β-1,4) marine xylan. Kinetic parameters, obtained using high-performance anion-exchange chromatography with pulsed amperometric detection and 3,5-dinitrosalicyclic acid reducing-sugar assays, show that TtGH8 catalyses the hydrolysis of β-1,4-xylohexaose with a kcat/Km of 7.5 × 107 M−1 min−1 but displays maximal activity against mixed-linkage polymeric xylans, hinting at a primary role in the degradation of marine polysaccharides. The three-dimensional structure of TtGH8 was solved in uncomplexed and xylobiose-, xylotriose- and xylohexaose-bound forms at approximately 1.5 Å resolution; the latter was consistent with the greater kcat/Km for hexasaccharide substrates. A 2,5B boat conformation observed in the −1 position of bound xylotriose is consistent with the proposed conformational itinerary for this class of enzyme. This work shows TtGH8 to be effective at the degradation of xylan-based substrates, notably marine xylan, further exemplifying the potential of T. turnerae for effective and diverse biomass degradation.
|
Oct 2018
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[7864, 9948]
Open Access
Abstract: A dominant human gut microbe, the well studied symbiont Bacteroides thetaiotaomicron (Bt), is a glyco-specialist that harbors a large repertoire of genes devoted to carbohydrate processing. Despite strong similarities among them, many of the encoded enzymes have evolved distinct substrate specificities, and through the clustering of cognate genes within operons termed polysaccharide-utilization loci (PULs) enable the fulfilment of complex biological roles. Structural analyses of two glycoside hydrolase family 92 α-mannosidases, BT3130 and BT3965, together with mechanistically relevant complexes at 1.8–2.5 Å resolution reveal conservation of the global enzyme fold and core catalytic apparatus despite different linkage specificities. Structure comparison shows that Bt differentiates the activity of these enzymes through evolution of a highly variable substrate-binding region immediately adjacent to the active site. These observations unveil a genetic/biochemical mechanism through which polysaccharide-processing bacteria can evolve new and specific biochemical activities from otherwise highly similar gene products.
|
May 2018
|
|
|
|
Andriy
Kryshtafovych
,
Reinhard
Albrecht
,
Arnaud
Basle
,
Pedro
Bule
,
Alessandro T.
Caputo
,
Ana Luisa
Carvalho
,
Kinlin L.
Chao
,
Ron
Diskin
,
Krzysztof
Fidelis
,
Carlos M. G. A.
Fontes
,
Folmer
Fredslund
,
Harry J.
Gilbert
,
Celia W.
Goulding
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Osnat
Herzberg
,
Johan C.
Hill
,
Andrzej
Joachimiak
,
Gert-wieland
Kohring
,
Roman I.
Koning
,
Leila
Lo Leggio
,
Marco
Mangiagalli
,
Karolina
Michalska
,
John
Moult
,
Shabir
Najmudin
,
Marco
Nardini
,
Valentina
Nardone
,
Didier
Ndeh
,
Thanh-hong
Nguyen
,
Guido
Pintacuda
,
Sandra
Postel
,
Mark J.
Van Raaij
,
Pietro
Roversi
,
Amir
Shimon
,
Abhimanyu K.
Singh
,
Eric J.
Sundberg
,
Kaspars
Tars
,
Nicole
Zitzmann
,
Torsten
Schwede
Abstract: The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.
|
Mar 2018
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[8425]
Abstract: The cellulosome is a remarkably intricate multienzyme nanomachine produced by anaerobic bacteria to degrade plant cell wall polysaccharides. Cellulosome assembly is mediated through binding of enzyme-borne dockerin modules to cohesin modules of the primary scaffoldin subunit. The anaerobic bacterium Acetivibrio cellulolyticus produces a highly intricate cellulosome comprising an adaptor scaffoldin, ScaB, whose cohesins interact with the dockerin of the primary scaffoldin (ScaA) that integrates the cellulosomal enzymes. The ScaB dockerin selectively binds to cohesin modules in ScaC that anchors the cellulosome onto the cell surface. Correct cellulosome assembly requires distinct specificities displayed by structurally related type I cohesin-dockerin pairs that mediate ScaC-ScaB and ScaA-enzyme assemblies. To explore the mechanism by which these two critical protein interactions display their required specificities, we determined the crystal structure of the dockerin of a cellulosomal enzyme in complex with a ScaA cohesin. The data revealed that the enzyme-borne dockerin binds to the ScaA cohesin in two orientations, indicating two identical cohesin-binding sites. Combined mutagenesis experiments served to identify amino acid residues that modulate type I cohesin-dockerin specificity in A. cellulolyticus. Rational design was used to test the hypothesis that the ligand-binding surfaces of ScaA- and ScaB-associated dockerins mediate cohesin recognition, independent of the structural scaffold. Novel specificities could thus be engineered into one, but not both of the ligand-binding sites of ScaB, while attempts at manipulating the specificity of the enzyme-associated dockerin were unsuccessful. These data indicate that dockerin specificity requires critical interplay between the ligand-binding surface and the structural scaffold of these modules.
|
Jan 2018
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
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.
|
Dec 2017
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
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]
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
|
Diamond Proposal Number(s):
[9948, 13587]
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
|
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.
|
May 2017
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
|
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
|
|
