Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

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.

Corrigendum: Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

This corrects the article DOI: 10.1038/nature21725.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition.

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 CBM families that targeted ß-glucans, ß-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 ß-glucans and ß-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.

Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and ß-1,3-1,4-Glucans through Distinct Mechanisms.

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with ß-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a ß-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to ß-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to ß-1,3-1,4-glucans while substantially decreasing the affinity for decorated ß-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified ß-1,3-1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant.

Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan.

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 ß-glucan chains often display broad specificity recognizing ß1,4-glucans (cellulose), ß1,3-ß1,4-mixed linked glucans and xyloglucan, a ß1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the ß1,4-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 ß-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a ß-sandwich fold. The ligand binding site comprises the ß-sheet that forms the concave surface of the proteins. Binding to the backbone chains of ß-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 ß-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln(106) is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which ß-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 ß-glucans.

Overproduction, purification, crystallization and preliminary X-ray characterization of the C-terminal family 65 carbohydrate-binding module (CBM65B) of endoglucanase Cel5A from Eubacterium cellulosolvens.

The rumen anaerobic cellulolytic bacterium Eubacterium cellulosolvens produces a large range of cellulases and hemicellulases responsible for the efficient hydrolysis of plant cell wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises a tandemly repeated carbohydrate-binding module (CBM65) fused to a glycoside hydrolase family 5 (Cel5A) catalytic domain, joined by flexible linker sequences. The second carbohydrate-binding module located at the C-terminus side of the endoglucanase (CBM65B) has been co-crystallized with either cellohexaose or xyloglucan heptasaccharide. The crystals belong to the hexagonal space group P6(5) and tetragonal space group P4(3)2(1)2, containing a single molecule in the asymmetric unit. The structures of CBM65B have been solved by molecular replacement.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum.

The modular carbohydrate-active enzyme belonging to glycoside hydrolase family 30 (GH30) from Clostridium thermocellum (CtXynGH30) is a cellulosomal protein which plays an important role in plant cell-wall degradation. The full-length CtXynGH30 contains an N-terminal catalytic module (Xyn30A) followed by a family 6 carbohydrate-binding module (CBM6) and a dockerin at the C-terminus. The recombinant protein has a molecular mass of 45 kDa. Preliminary structural characterization was carried out on Xyn30A crystallized in different conditions. All tested crystals belonged to space group P1 with one molecule in the asymmetric unit. Molecular replacement has been used to solve the Xyn30A structure.

The Jo-In protein welding system is a relevant tool to create CBM-containing plant cell wall degrading enzymes.

Irrespective of their biological origin, most proteins are composed of several elementary domains connected by linkers. These domains are either functionally independent units, or part of larger multidomain structures whose functions are defined by their spatial proximity. Carbohydrate-degrading enzymes provide examples of a range of multidomain structures, in which catalytic protein domains are frequently appended to one or more non-catalytic carbohydrate-binding modules which specifically bind to carbohydrate motifs. While the carbohydrate-binding specificity of these modules is clear, their function is not fully elucidated. Herein, an original approach to tackle the study of carbohydrate-binding modules using the Jo-In biomolecular welding protein pair is presented. To provide a proof of concept, recombinant xylanases appended to two different carbohydrate-binding modules have been created and produced. The data reveal the biochemical properties of four xylanase variants and provide the basis for correlating enzyme activity to structural properties and to the nature of the substrate and the ligand specificity of the appended carbohydrate-binding module. It reveals that specific spatial arrangements favour activity on soluble polymeric substrates and that activity on such substrates does not predict the behaviour of multimodular enzymes on insoluble plant cell wall samples. The results highlight that the Jo-In protein welding system is extremely useful to design multimodular enzyme systems, especially to create rigid conformations that decrease the risk of intermodular interference. Further work on Jo-In will target the introduction of varying degrees of flexibility, providing the means to study this property and the way it may influence multimodular enzyme functions.

Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides.

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.

Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome.

Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connected via linker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium, Ruminococcus flavefaciens strain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Šresolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using the CCP4 program suite and SHELXC/D/E.

Expression, purification, crystallization and preliminary X-ray analysis of CttA, a putative cellulose-binding protein from Ruminococcus flavefaciens.

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 P3(1)21 or P3(2)21 and P2(1), respectively. The structure was phased using bound iodide from the crystallization buffer by SAD experiments.

Purification and crystallographic studies of a putative carbohydrate-binding module from the Ruminococcus flavefaciens FD-1 endoglucanase Cel5A.

Ruminant herbivores meet their carbon and energy requirements from a symbiotic relationship with cellulosome-producing anaerobic bacteria that efficiently degrade plant cell-wall polysaccharides. The assembly of carbohydrate-active enzymes (CAZymes) into cellulosomes enhances protein stability and enzyme synergistic interactions. Cellulosomes comprise diverse CAZymes displaying a modular architecture in which a catalytic domain is connected, via linker sequences, to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus facilitating catalysis. The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD-1 contains over 200 modular proteins containing the cellulosomal signature dockerin module. One of these is an endoglucanase Cel5A comprising two family 5 glycoside hydrolase catalytic modules (GH5) flanking an unclassified CBM (termed CBM-Rf2) and a C-terminal dockerin. This novel CBM-Rf2 has been purified and crystallized, and data from cacodylate-derivative crystals were processed to 1.02 and 1.29 Šresolution. The crystals belonged to the orthorhombic space group P212121. The CBM-Rf2 structure was solved by a single-wavelength anomalous dispersion experiment at the As edge.

Overproduction, purification, crystallization and preliminary X-ray characterization of the family 46 carbohydrate-binding module (CBM46) of endo-ß-1,4-glucanase B (CelB) from Bacillus halodurans.

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-ß-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 Šresolution, respectively. The BhCBM46 structure was determined by a single-wavelength anomalous dispersion experiment using AutoSol from the PHENIX suite.

Crystallization and preliminary X-ray diffraction analysis of a trimodular endo-ß-1,4-glucanase (Cel5B) from Bacillus halodurans.

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-ß-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.

Expression, purification and crystallization of a novel carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome.

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.