Xyloglucan Utilization Research Articles

Crystal Structure of α-Xylosidase from Aspergillus niger in Complex with a Hydrolyzed Xyloglucan Product and New Insights in Accurately Predicting Substrate Specificities of GH31 Family Glycosidases.

Glycoside hydrolase family 31 (GH31) enzymes show both highly conserved folds and catalytic residues. Yet different members of GH31 show very different substrate specificities, and it is not obvious how these specificities arise from the protein sequences. The fungal α-xylosidase, AxlA, was originally isolated from a commercial enzyme mixture secreted by Aspergillus niger and was reported to have potential as a catalytic component in biomass deconstruction in the biofuel industry. We report here the crystal structure of AxlA in complex with its catalytic product, a hydrolyzed xyloglucan oligosaccharide. On the basis of our new structure, we provide the structural basis for AxlA’s role in xyloglucan utilization and, more importantly, a new procedure to predict and differentiate C5 vs C6 sugar specific activities based on protein sequences of the functionally diverse GH31 family enzymes.

Adaptation of Syntenic Xyloglucan Utilization Loci of Human Gut Bacteroidetes to Polysaccharide Side Chain Diversity.

Genome sequencing has revealed substantial variation in the predicted abilities of individual species within animal gut microbiota to metabolize the complex carbohydrates comprising dietary fiber. At the same time, a currently limited body of functional studies precludes a richer understanding of how dietary glycan structures affect the gut microbiota composition and community dynamics. Here, using biochemical and biophysical techniques, we identified and characterized differences among recombinant proteins from syntenic xyloglucan utilization loci (XyGUL) of three Bacteroides and one Dysgonomonas species from the human gut, which drive substrate specificity and access to distinct polysaccharide side chains. Enzymology of four syntenic glycoside hydrolase family 5 subfamily 4 (GH5_4) endo-xyloglucanases revealed surprising differences in xyloglucan (XyG) backbone cleavage specificity, including the ability of some homologs to hydrolyze congested branched positions. Further, differences in the complement of GH43 alpha-l-arabinofuranosidases and GH95 alpha-l-fucosidases among syntenic XyGUL confer distinct abilities to fully saccharify plant species-specific arabinogalactoxyloglucan and/or fucogalactoxyloglucan. Finally, characterization of highly sequence-divergent cell surface glycan-binding proteins (SGBPs) across syntenic XyGUL revealed a novel group of XyG oligosaccharide-specific SGBPs encoded within select BacteroidesIMPORTANCE The catabolism of complex carbohydrates that otherwise escape the endogenous digestive enzymes of humans and other animals drives the composition and function of the gut microbiota. Thus, detailed molecular characterization of dietary glycan utilization systems is essential both to understand the ecology of these complex communities and to manipulate their compositions, e.g., to benefit human health. Our research reveals new insight into how ubiquitous members of the human gut microbiota have evolved a set of microheterogeneous gene clusters to efficiently respond to the structural variations of plant xyloglucans. The data here will enable refined functional prediction of xyloglucan utilization among diverse environmental taxa in animal guts and beyond.

A Cell-Surface GH9 Endo-Glucanase Coordinates with Surface Glycan-Binding Proteins to Mediate Xyloglucan Uptake in the Gut Symbiont Bacteroides ovatus

Dietary fiber is an important food source for members of the human gut microbiome. Members of the dominant Bacteroidetes phylum capture diverse polysaccharides via the action of multiple cell surface proteins encoded within polysaccharide utilization loci (PUL). The independent activities of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) for the harvest of various glycans have been studied in detail, but how these proteins work together to coordinate uptake is poorly understood. Here, we combine genetic and biochemical approaches to discern the interplay between the BoGH9 endoglucanase and the xyloglucan-binding proteins SGBP-A and SGBP-B from the Bacteroides ovatus xyloglucan utilization locus (XyGUL). The expression of BoGH9, a weakly active xyloglucanase in isolation, is required in a strain that expresses a non-binding version of SGBP-A (SGBP-A*). The crystal structure of the BoGH9 enzyme suggests the molecular basis for its robust activity on mixed-linkage β-glucan compared to xyloglucan. However, catalytically inactive site-directed mutants of BoGH9 fail to complement the deletion of the active BoGH9 in a SGBP-A* strain. We also find that SGBP-B is needed in an SGBP-A* background to support growth on xyloglucan, but that the non-binding SGBP-B* protein acts in a dominant negative manner to inhibit growth on xyloglucan. We postulate a model whereby the SGBP-A, SGBP-B, and BoGH9 work together at the cell surface, likely within a discrete complex, and that xyloglucan binding by SGBP-B and BoGH9 may facilitate the orientation of the xyloglucan for transfer across the outer membrane.

In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions

BackgroundXyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved.ResultsCombined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here.ConclusionsThrough a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural–functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.

Delayed utilization of some fast-fermenting soluble dietary fibers by human gut microbiota when presented in a mixture

Delivering fibers more distally could be important to prevent or treat colonic diseases such as cancer and ulcerative colitis. Here, we hypothesized that fermentation of fast-fermenting soluble fibers by the colonic microbiota is delayed when they are presented in a mixture due to hierarchical utilization of fibers. A series of in vitro fermentation studies was performed using fecal microbiota obtained from three healthy donors using single dietary fibers [arabinoxylan, chondroitin sulfate (CS), galactomannan (GM), polygalacturonic acid (PGA), xyloglucan (XG)] and a mixture containing an equal amount of each. Substrate disappearance analysis, as measured by GC–MS, revealed that CS, PGA, and XG utilization was delayed when present in the mixture. 16S rRNA sequencing showed certain fibers consistently increased specific genera in the microbiota of all donor groups. Mixing different types of fermentable dietary fibers might be a logical strategy for delivering fibers into more distal regions of the colon.

Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut.

The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.

Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont.

ABSTRACTPolysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes.

Functional and structural characterization of a potent GH74 endo-xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation.

The heteropolysaccharide xyloglucan (XyG) comprises up to one-quarter of the total carbohydrate content of terrestrial plant cell walls and, as such, represents a significant reservoir in the global carbon cycle. The complex composition of XyG requires a consortium of backbone-cleaving endo-xyloglucanases and side-chain cleaving exo-glycosidases for complete saccharification. The biochemical basis for XyG utilization by the model Gram-negative soil saprophytic bacterium Cellvibrio japonicus is incompletely understood, despite the recent characterization of associated side-chain cleaving exo-glycosidases. We present a detailed functional and structural characterization of a multimodular enzyme encoded by gene locus CJA_2477. The CJA_2477 gene product comprises an N-terminal glycoside hydrolase family 74 (GH74) endo-xyloglucanase module in train with two carbohydrate-binding modules (CBMs) from families 10 and 2 (CBM10 and CBM2). The GH74 catalytic domain generates Glc4 -based xylogluco-oligosaccharide (XyGO) substrates for downstream enzymes through an endo-dissociative mode of action. X-ray crystallography of the GH74 module, alone and in complex with XyGO products spanning the entire active site, revealed a broad substrate-binding cleft specifically adapted to XyG recognition, which is composed of two seven-bladed propeller domains characteristic of the GH74 family. The appended CBM10 and CBM2 members notably did not bind XyG, nor other soluble polysaccharides, and instead were specific cellulose-binding modules. Taken together, these data shed light on the first step of xyloglucan utilization by C. japonicus and expand the repertoire of GHs and CBMs for selective biomass analysis and utilization. Structural data have been deposited in the RCSB protein database under the Protein Data Bank codes: 5FKR, 5FKS, 5FKT and 5FKQ.

How Gut Bacteroides Eat Your Veggies

The human gut Bacteroidetes display a greatly expanded capacity for glycan degradation via the expression of multiple outermembrane protein complexes, termed Sus‐like systems. These systems are comprised of proteins that sequentially bind, degrade and import specific carbohydrates. Two classes of carbohydrate‐binding proteins, the SusD‐like proteins and the carbohydrate‐binding lipoproteins (CBLPs) respectively, are functionally conserved within the Bacteroidetes Sus‐like systems and found in all systems. We have recently determined the crystal structures two such cell surface liopoproteins, XgusD, a SusD‐like protein, and XgusE, a CBLP, from Bacteroides ovatus. These proteins are involved in the recognition of xyloglucan, a plant cell wall polysaccharide abundant in many “salad” vegetables including lettuce, tomato and peppers. Xyloglucan is comprised of a beta‐glucan backbone appended with xylose and galactose side chains, and we demonstrate that recognition of this polymer by the XgusD and XgusE proteins is driven by different aspects of the carbohydrate structure. More over, the XgusE protein displays a novel xyloglucan‐binding domain, and likely reflects a new class of carbohydrate‐binding modules. While XgusD is essential for all xyloglucan utilization, mutants of XgusE in B. ovatus are impaired in the uptake of some xyloglucan oligosaccharides. Taken together our data support our previous work demonstrating that the SusD‐like proteins and CBLPs have evolved to play non‐redundant roles in carbohydrate uptake.

A complex gene locus enables xyloglucan utilization in the model saprophyte Cellvibrio japonicus.

The degradation of plant biomass by saprophytes is an ecologically important part of the global carbon cycle, which has also inspired a vast diversity of industrial enzyme applications. The xyloglucans (XyGs) constitute a family of ubiquitous and abundant plant cell wall polysaccharides, yet the enzymology of XyG saccharification is poorly studied. Here, we present the identification and molecular characterization of a complex genetic locus that is required for xyloglucan utilization by the model saprophyte Cellvibrio japonicus. In harness, transcriptomics, reverse genetics, enzyme kinetics, and structural biology indicate that the encoded cohort of an α-xylosidase, a β-galactosidase, and an α-l-fucosidase is specifically adapted for efficient, concerted saccharification of dicot (fucogalacto)xyloglucan oligosaccharides following import into the periplasm via an associated TonB-dependent receptor. The data support a biological model of xyloglucan degradation by C. japonicus with striking similarities – and notable differences – to the complex polysaccharide utilization loci of the Bacteroidetes.

A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes.

A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed “dietary fibre,” from the cell walls of diverse fruits and vegetables.1 Due to a paucity of alimentary enzymes encoded by the human genome,2 our ability to derive energy from dietary fibre depends on saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut.3,4 The xyloglucans (XyGs), in particular, are a ubiquitous family of highly branched plant cell wall polysaccharides5,6 whose mechanism(s) of degradation in the human gut and consequent importance in nutrition was heretofore unknown.1,7,8 Here, we demonstrate that a single, complex gene locus in Bacteroides ovatus confers xyloglucan catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous xyloglucan utilization loci (XyGULs) serve as genetic markers of xyloglucan catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health.9–12

Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification

The desire for improved methods of biomass conversion into fuels and feedstocks has re-awakened interest in the enzymology of plant cell wall degradation. The complex polysaccharide xyloglucan is abundant in plant matter, where it may account for up to 20% of the total primary cell wall carbohydrates. Despite this, few studies have focused on xyloglucan saccharification, which requires a consortium of enzymes including endo-xyloglucanases, α-xylosidases, β-galactosidases and α-L-fucosidases, among others. In the present paper, we show the characterization of Xyl31A, a key α-xylosidase in xyloglucan utilization by the model Gram-negative soil saprophyte Cellvibrio japonicus. CjXyl31A exhibits high regiospecificity for the hydrolysis of XGOs (xylogluco-oligosaccharides), with a particular preference for longer substrates. Crystallographic structures of both the apo enzyme and the trapped covalent 5-fluoro-β-xylosyl-enzyme intermediate, together with docking studies with the XXXG heptasaccharide, revealed, for the first time in GH31 (glycoside hydrolase family 31), the importance of a PA14 domain insert in the recognition of longer oligosaccharides by extension of the active-site pocket. The observation that CjXyl31A was localized to the outer membrane provided support for a biological model of xyloglucan utilization by C. japonicus, in which XGOs generated by the action of a secreted endo-xyloglucanase are ultimately degraded in close proximity to the cell surface. Moreover, the present study diversifies the toolbox of glycosidases for the specific modification and saccharification of cell wall polymers for biotechnological applications.