The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome.

The Bacteroides thetaiotaomicron has developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical ß-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

Feasibility of Brewer's Spent Yeast Microcapsules as Targeted Oral Carriers.

Brewer's spent yeast (BSY) microcapsules have a complex network of cell-wall polysaccharides that are induced by brewing when compared to the baker's yeast (Saccharomyces cerevisiae) microcapsules. These are rich in (ß1→3)-glucans and covalently linked to (α1→4)- and (ß1→4)-glucans in addition to residual mannoproteins. S. cerevisiae is often used as a drug delivery system due to its immunostimulatory potential conferred by the presence of (ß1→3)-glucans. Similarly, BSY microcapsules could also be used in the encapsulation of compounds or drug delivery systems with the advantage of resisting digestion conferred by (ß1→4)-glucans and promoting a broader immunomodulatory response. This work aims to study the feasibility of BSY microcapsules that are the result of alkali and subcritical water extraction processes, as oral carriers for food and biomedical applications by (1) evaluating the resistance of BSY microcapsules to in vitro digestion (IVD), (2) their recognition by the human Dectin-1 immune receptor after IVD, and (3) the recognition of IVD-solubilized material by different mammalian immune receptors. IVD digested 44-63% of the material, depending on the extraction process. The non-digested material, despite some visible agglutination and deformation of the microcapsules, preserved their spherical shape and was enriched in (ß1→3)-glucans. These microcapsules were all recognized by the human Dectin-1 immune receptor. The digested material was differentially recognized by a variety of lectins of the immune system related to (ß1→3)-glucans, glycogen, and mannans. These results show the potential of BSY microcapsules to be used as oral carriers for food and biomedical applications.

Structural differences on cell wall polysaccharides of brewer's spent Saccharomyces and microarray binding profiles with immune receptors.

Brewing practice uses the same yeast to inoculate the following fermentation (repitching). Saccharomyces pastorianus, used to produce Lager beer, is widely reused, not changing its fermentation performance. However, S. cerevisiae, used to produce Ale beer, is partial or not even reused, due to its poor performance. It is hypothesized that cells modulate their wall polysaccharides to increase the cell-wall strength. In this work industrial S. cerevisiae and S. pastorianus brewer's spent yeasts with different repitching numbers were studied. Glucans were the main component of S. cerevisiae whereas mannoproteins were abundant in S. pastorianus. The major changes were noticed on glucans of both species, ß1,3-glucans decrease more pronounced in S. cerevisiae. The increase of α1,4-Glc, related with osmotolerance, was higher in S. cerevisiae while ß1,4-Glc, related with cell-wall strength, had a small increase. In addition, these structural details showed different binding profiles to immune receptors, important to develop tailored bioactive applications.

Mapping Molecular Recognition of ß1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus.

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage ß1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a ß1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward ß1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of ß1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial ß1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the ß-glucan backbone imposed by the ß1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long ß1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of ß1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage ß1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.

Structural analysis and potential immunostimulatory activity of Nannochloropsis oculata polysaccharides.

The relevance of microalgae biotechnology for producing high-value compounds with biomedical application, such as polysaccharides, has been increasing. Despite this, the knowledge about the composition and structure of microalgae polysaccharides is still scarce. In this work, water-soluble polysaccharides from Nannochloropsis oculata were extracted, fractionated, structurally analysed, and subsequently tested in terms of immunostimulatory activity. A combination of sugar and methylation analysis with interaction data of carbohydrate-binding proteins using carbohydrate microarrays disclosed the complex structural features of the different polysaccharides. These analyses showed that the water-soluble polysaccharides fractions from N. oculata were rich in (ß1→3, ß1→4)-glucans, (α1→3)-, (α1→4)-mannans, and anionic sulphated heterorhamnans. The immunostimulatory assay highlighted that these fractions could also stimulate murine B-lymphocytes. Thus, the N. oculata water-soluble polysaccharides show potential to be further explored for immune-mediated biomedical applications.

Diverse specificity of cellulosome attachment to the bacterial cell surface.

During the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

Putting an N-terminal end to the Clostridium thermocellum xylanase Xyn10B story: crystal structure of the CBM22-1-GH10 modules complexed with xylohexaose.

In general, plant cell wall degrading enzymes are modular proteins containing catalytic domains linked to one or more non-catalytic carbohydrate-binding modules (CBMs). Xyn10B from Clostridium thermocellum is a typical modular enzyme containing an N-terminal family 22 CBM (CBM22-1), a family 10 glycoside hydrolase catalytic domain (GH10), a second CBM22 (CBM22-2), a dockerin sequence and a C-terminal family 1 carbohydrate esterase (CE1) catalytic domain. The structure of the N-terminal bi-modular CBM22-1-GH10 component of Xyn10B has been determined using a SeMet derivative by SAD to 2.5Å. The data was extended to 2.0Å for the non-SeMet mutant complexed with xylohexaose. CBM22-1-GH10 is a 60kDa protein with an E337A mutation to render the GH10 subunit inactive. Three of the six xylose residues of xylohexaose are shown to be bound in the inactivated GH10 substrate binding cleft, with the other three sugars presumably disordered in the solvent channel. The protein is a dimer in the asymmetric unit with extensive surface contacts between the two GH10 modules and between the CBM22-1 and GH10 modules. Residues from helix H4 of the GH10 module provide the major contacts by fitting into the minor groove of the CBM22-1 module. The orientation of CBM22-1 is such that it would allow the substrate to be loosely bound and subsequently delivered to the active site in a processive manner.

Functional insights into the role of novel type I cohesin and dockerin domains from Clostridium thermocellum.

Cellulosomes, synthesized by anaerobic microorganisms such as Clostridium thermocellum, are remarkably complex nanomachines that efficiently degrade plant cell wall polysaccharides. Cellulosome assembly results from the interaction of type I dockerin domains, present on the catalytic subunits, and the cohesin domains of a large non-catalytic integrating protein that acts as a molecular scaffold. In general, type I dockerins contain two distinct cohesin-binding interfaces that appear to display identical ligand specificities. Inspection of the C. thermocellum genome reveals 72 dockerin-containing proteins. In four of these proteins, Cthe_0258, Cthe_0435, Cthe_0624 and Cthe_0918, there are significant differences in the residues that comprise the two cohesin-binding sites of the type I dockerin domains. In addition, a protein of unknown function (Cthe_0452), containing a C-terminal cohesin highly similar to the equivalent domains present in C. thermocellum-integrating protein (CipA), was also identified. In the present study, the ligand specificities of the newly identified cohesin and dockerin domains are described. The results revealed that Cthe_0452 is located at the C. thermocellum cell surface and thus the protein was renamed as OlpC. The dockerins of Cthe_0258 and Cthe_0435 recognize, preferentially, the OlpC cohesin and thus these enzymes are believed to be predominantly located at the surface of the bacterium. By contrast, the dockerin domains of Cthe_0624 and Cthe_0918 are primarily cellulosomal since they bind preferentially to the cohesins of CipA. OlpC, which is a relatively abundant protein, may also adopt a 'warehouse' function by transiently retaining cellulosomal enzymes at the cell surface before they are assembled on to the multienzyme complex.

The active site of a carbohydrate esterase displays divergent catalytic and noncatalytic binding functions.

Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of "gene sharing," where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.

Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-beta-D-xylanase 10B in complex with xylohexaose.

The cellulosome of Clostridium thermocellum is a highly organized multi-enzyme complex of cellulases and hemicellulases involved in the hydrolysis of plant cell-wall polysaccharides. The bifunctional multi-modular xylanase Xyn10B is one of the hemicellulase components of the C. thermocellum cellulosome. The enzyme contains an internal glycoside hydrolase family 10 catalytic domain (GH10) and a C-terminal family 1 carbohydrate esterase domain (CE1). The N-terminal moiety of Xyn10B (residues 32-551), comprising a carbohydrate-binding module (CBM22-1) and the GH10 E337A mutant, was crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3(2)21 and contain a dimer in the asymmetric unit. The crystals diffracted to beyond 2.0 A resolution.

The Clostridium cellulolyticum dockerin displays a dual binding mode for its cohesin partner.

The plant cell wall degrading apparatus of anaerobic bacteria includes a large multienzyme complex termed the "cellulosome." The complex assembles through the interaction of enzyme-derived dockerin modules with the multiple cohesin modules of the noncatalytic scaffolding protein. Here we report the crystal structure of the Clostridium cellulolyticum cohesin-dockerin complex in two distinct orientations. The data show that the dockerin displays structural symmetry reflected by the presence of two essentially identical cohesin binding surfaces. In one binding mode, visualized through the A16S/L17T dockerin mutant, the C-terminal helix makes extensive interactions with its cohesin partner. In the other binding mode observed through the A47S/F48T dockerin variant, the dockerin is reoriented by 180 degrees and interacts with the cohesin primarily through the N-terminal helix. Apolar interactions dominate cohesin-dockerin recognition that is centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied, in the two binding modes, by the dockerin residues Phe-19 and Leu-50, respectively. Despite the structural similarity between the C. cellulolyticum and Clostridium thermocellum cohesins and dockerins, there is no cross-specificity between the protein partners from the two organisms. The crystal structure of the C. cellulolyticum complex shows that organism-specific recognition between the protomers is dictated by apolar interactions primarily between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. The biological significance of the plasticity in dockerin-cohesin recognition, observed here in C. cellulolyticum and reported previously in C. thermocellum, is discussed.