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

Abstract

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 Mechanism by which Prominent Human Gut Bacteroidetes Utilize Mixed-Linkage Beta-Glucans, Major Health-Promoting Cereal Polysaccharides

“…However, BoGH3 MLG has 63% sequence identity to a B. ovatus β-glucosidase (BoGH3B, PDB code 5JP0 (Hemsworth et al, 2016)), from the xyloglucan utilization locus (Fig. S5A) and, as such, was amenable to tertiary structure homology modelling.…”

“…Analogous to the archetypal Bacteroides thetaiotaomicron starch utilization system (Sus), a hallmark of all Bacteroidetes PULs is the organization of genes clustered around tandem susC / susD homologs (encoding a TonB dependent transporter, TBDT, and a cell-surface glycan-binding protein, SGBP, respectively) (Martens et al, 2009). Additional co-localized and co-regulated SGBP(s), a cohort of enzymes, and a transcriptional regulator typically make up a machinery that acts in concert to sense, break down, and import complex glycans (Grondin et al, 2017; Hemsworth et al, 2016). Many such PULs, each targeting specific glycan structures, have been identified by genomics and transcriptomics (see, e.g.…”

“…Recently, comprehensive functional analysis has revealed the detailed molecular mechanisms by which individual PULs enable human gut Bacteroidetes to utilize predominant plant polysaccharides, including the matrix glycans, xyloglucan (Hemsworth et al, 2016; Larsbrink et al, 2014; Tauzin et al, 2016), xylan (Rogowski et al, 2015), β-mannan (Bågenholm et al, 2017), and rhamnogalacturonan II (Ndeh et al, 2017). Mixed-linkage β(1,3)/β(1,4)-glucans (MLGs, Fig.…”

Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

“…The colocalization of Bgl3D with the sidechain-cleaving exo-glycosidases (Xyl31A, Bgl35A and Afc95A) of the XyGUL in the periplasm of C. japonicus gives evidence of a unified strategy for competitive oligosaccharide acquisition and utilization by this saprophytic bacterium. This elegant system is analogous to that used by the human gut symbiont Bacterioides ovatus, in which two periplasmic GH3s operate in concert with an a-xylosidase, a b-galactosidase and two a-L-arabinofuranosidases to saccharify dietary (arabinogalacto)xyloglucans (Larsbrink et al, 2014b;Hemsworth et al, 2016).…”

Polysaccharide Utilization Loci: Fueling Microbial Communities

“…1) (78). The saccharification pathway of this ubiquitous, highly branched dietary glycan was determined through the biochemical and structural characterization of all eight GHs and two SGBPs from the XyGUL, in harness with reverse genetics (72,78,109). These studies were instrumental in highlighting the adaptive evolution of GH cohorts among syntenic XyGULs.…”