How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans

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

Crystallization and preliminary X-ray analysis of RsbS from Moorella thermoacetica at 2.5 Å resolution

Crystallization and selenium substructure solution of RsbS from Moorella thermoacetica, the first ab initio phased crystal structure from Diamond

Investigating the Role of Zinc and Copper Binding Motifs of Trafficking Sites in the Cyanobacterium Synechocystis PCC 6803

Although zinc and copper are required by proteins with very different functions, these metals can be delivered to cellular locations by homologous metal transporters within the same organism, as demonstrated by the cyanobacterial (Synechocystis PCC 6803) zinc exporter ZiaA and thylakoidal copper importer PacS. The N-terminal metal-binding domains of these transporters (ZiaA_N and PacS_N, respectively) have related ferredoxin folds also found in the metallochaperone Atx1, which delivers copper to PacS, but differ in the residues found in their M/IXCXXC metal-binding motifs. To investigate the role of the nonconserved residues in this region on metal binding, the sequence from ZiaA_N has been introduced into Atx1 and PacS_N, and the motifs of Atx1 and PacS_N swapped. The motif sequence can tune Cu­(I) affinity only approximately 3-fold. However, the introduction of the ZiaA_N motif (MDCTSC) dramatically increases the Zn­(II) affinity of both Atx1 and PacS_N by up to 2 orders of magnitude. The Atx1 mutant with the ZiaA_N motif crystallizes as a side-to-side homodimer very similar to that found for [Cu­(I)₂–Atx1]₂ (Badarau et al. Biochemistry 2010, 49, 7798). In a crystal structure of the PacS_N mutant possessing the ZiaA_N motif (PacS_N^ZiaA_N), the Asp residue from the metal-binding motif coordinates Zn­(II). This demonstrates that the increased Zn­(II) affinity of this variant and the high Zn­(II) affinity of ZiaA_N are due to the ability of the carboxylate to ligate this metal ion. Comparison of the Zn­(II) sites in PacS_N^ZiaA_N structures provides additional insight into Zn­(II) trafficking in cyanobacteria

Molecular architecture of the 'stressosome,' a signal integration and transduction hub

A commonly used strategy by microorganisms to survive multiple stresses involves a signal transduction cascade that increases the expression of stress-responsive genes. Stress signals can be integrated by a multiprotein signaling hub that responds to various signals to effect a single outcome. We obtained a medium- resolution cryo- electron microscopy reconstruction of the 1.8- megadalton "stressosome" from Bacillus subtilis. Fitting known crystal structures of components into this reconstruction gave a pseudoatomic structure, which had a virus capsid-like core with sensory extensions. We suggest that the different sensory extensions respond to different signals, whereas the conserved domains in the core integrate the varied signals. The architecture of the stressosome provides the potential for cooperativity, suggesting that the response could be tuned dependent on the magnitude of chemophysical insult

Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-beta-D-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or Delta 4,5-anhydrogalaturonic acid (Delta 4,5-GalA). Delta 4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.</p