Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.

The Echinococcus granulosus actin filament-fragmenting protein (EgAFFP) is a three domain member of the gelsolin family of proteins, which is antigenic to human hosts. These proteins, formed by three or six conserved domains, are involved in the dynamic rearrangements of the cytoskeleton, being responsible for severing and capping actin filaments and promoting nucleation of actin monomers. Various structures of six domain gelsolin-related proteins have been investigated, but little information on the structure of three domain members is available. In this work, the solution structure of the three domain EgAFFP has been investigated through small-angle x-ray scattering (SAXS) studies. EgAFFP exhibits an elongated molecular shape. The radius of gyration and the maximum dimension obtained by SAXS were, respectively, 2.52 +/- 0.01 nm and 8.00 +/- 1.00 nm, both in the absence and presence of Ca2+. Two different molecular homology models were built for EgAFFP, but only one was validated through SAXS studies. The predicted structure for EgAFFP consists of three repeats of a central beta-sheet sandwiched between one short and one long alpha-helix. Possible implications of the structure of EgAFFP upon actin binding are discussed.

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Å resolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite -1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.

Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is α-D-glucuronic acid, which is linked to the O2' position of the β-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific α-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of α-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65 Å resolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The xylohexaose moieties of the xylohexaose are recognized by residues from both protomers, thus creating a xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca2+ ions were identified in the wtsAgu115A structures. One Ca2+ ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg2+ or Mn2+ resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca2+ resulted in a decrease in activity.

Using globally abundant crop residues as a carbon source for energy generation and renewable chemicals production stand out as a promising solution to reduce current dependency on fossil fuels. In nature, such as in compost habitats, microbial communities efficiently degrade the available plant biomass using a diverse set of synergistic enzymes. However, deconstruction of lignocellulose remains a challenge for industry due to recalcitrant nature of the substrate and the inefficiency of the enzyme systems available, making the economic production of lignocellulosic biofuels difficult. Metatranscriptomic studies of microbial communities can unveil the metabolic functions employed by lignocellulolytic consortia and identify novel biocatalysts that could improve industrial lignocellulose conversion.

Lignocellulose is one of the most abundant forms of fixed carbon in the biosphere. Current industrial approaches to the degradation of lignocellulose employ enzyme mixtures, usually from a single fungal species, which are only effective in hydrolyzing polysaccharides following biomass pre-treatments. While the enzymatic mechanisms of lignocellulose degradation have been characterized in detail in individual microbial species, the microbial communities that efficiently breakdown plant materials in nature are species rich and secrete a myriad of enzymes to perform "community-level" metabolism of lignocellulose. Single-species approaches are, therefore, likely to miss important aspects of lignocellulose degradation that will be central to optimizing commercial processes.

Here the statistics concerning X-ray data processing and structure refinement are given, together with the substrate preference analysis for ThBgl1 and ThBgl2. Finally, the analysis of the influence of temperature and pH on the activities of both enzymes are shown.

Interleukin-22 (IL-22) plays an important role in the regulation of immune and inflammatory responses in mammals. The IL-22 binding protein (IL-22BP), a soluble receptor that specifically binds IL-22, prevents the IL-22/interleukin-22 receptor 1 (IL-22R1)/interleukin-10 receptor 2 (IL-10R2) complex assembly and blocks IL-22 biological activity. Here we present the crystal structure of the IL-22/IL-22BP complex at 2.75 A resolution. The structure reveals IL-22BP residues critical for IL-22 binding, which were confirmed by site-directed mutagenesis and functional studies. Comparison of IL-22/IL-22BP and IL-22/IL-22R1 crystal structures shows that both receptors display an overlapping IL-22 binding surface, which is consistent with the inhibitory role played by IL-22 binding protein.

Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endo-β-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from Paenibacillus xylanivorans A57 (PxXyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The PxXyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of PxXyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.

Liver X receptor (LXR) is an attractive drug target for the development of novel therapeutic agents for the treatment of dyslipidaemia and cholestasis. In the present work, comparative molecular field analysis (CoMFA) and hologram quantitative structure-activity relationship (HQSAR) studies were conducted on a series of potent LXR ligands. Significant correlation coefficients (CoMFA, r(2) = 0.98 and q(2) = 0.69; HQSAR, r(2) = 0.99 and q(2) = 0.85) were obtained, indicating the potential of the models for untested compounds. The models were then used to predict the potency of an external test set, and the predicted values obtained from the 2D and 3D models were in good agreement with the experimental results. The final QSAR models, along with the information obtained from 3D steric and electrostatic contour maps and 2D contribution maps should be useful for the design of novel LXR ligands having improved potency.

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. In the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. The sequencing generated 14.7 Gbp for downstream analyses. De novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. The present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

Peroxisome proliferator activated receptors (PPARs δ, α and γ) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.

Microbial communities (MCs) create complex metabolic networks in natural habitats and respond to environmental changes by shifts in the community structure. Although members of MCs are often not amenable for cultivation in pure culture, it is possible to obtain a greater diversity of species in the laboratory setting when microorganisms are grown as mixed cultures. In order to mimic the environmental conditions, an appropriate growth medium must be applied. Here, we examined the hypothesis that a greater diversity of microorganisms can be sustained under nutrient-limited conditions. Using a 16 S rRNA amplicon metagenomic approach, we explored the structure of a compost-derived MC. During a five-week time course the MC grown in minimal medium with sugarcane bagasse (SCB) as a sole carbon source showed greater diversity and enrichment in lignocellulose-degrading microorganisms. In contrast, a MC grown in nutrient rich medium with addition of SCB had a lower microbial diversity and limited number of lignocellulolytic species. Our approach provides evidence that factors such as nutrient availability has a significant selective pressure on the biodiversity of microorganisms in MCs. Consequently, nutrient-limited medium may displace bacterial generalist species, leading to an enriched source for mining novel enzymes for biotechnology applications.

Microbial communities metabolize plant biomass using secreted enzymes; however, identifying extracellular proteins tightly bound to insoluble lignocellulose in these microbiomes presents a challenge, as the rigorous extraction required to elute these proteins also lyses the microbes associated with the plant biomass releasing intracellular proteins that contaminate the metasecretome. Here we describe a technique for targeting the extracellular proteome, which was used to compare the metasecretome and meta-surface-proteome of two lignocellulose-degrading communities grown on wheat straw and rice straw. A combination of mass spectrometry-based proteomics coupled with metatranscriptomics enabled the identification of a unique secretome pool from these lignocellulose-degrading communities. This method enabled us to efficiently discriminate the extracellular proteins from the intracellular proteins by improving detection of actively secreted and transmembrane proteins. In addition to the expected carbohydrate active enzymes, our new method reveals a large number of unknown proteins, supporting the notion that there are major gaps in our understanding of how microbial communities degrade lignocellulosic substrates.

β-glucosidases are glycoside hydrolases able to cleave small and soluble substrates, thus producing monosaccharides. These enzymes are distributed among families GH1, GH2, GH3, GH5, GH9, GH30 and GH116, with GH1 and GH3 being the most relevant families with characterized enzymes to date. A recent transcriptomic analysis of the fungus Trichoderma harzianum, known for its increased β-glucosidase activity as compared to Trichoderma reesei, revealed two enzymes from family GH1 with high expression levels. Here we report the cloning, recombinant expression, purification and crystallization of these enzymes, ThBgl1 and ThBgl2. A close inspection of the enzymatic activity of these enzymes surprisingly revealed a marked difference between them despite the sequence similarity (53%). ThBgl1 has an increased tendency to catalyze transglycosylation reaction while ThBgl2 acts more as a hydrolyzing enzyme. Detailed comparison of their crystal structures and the analysis of the molecular dynamics simulations reveal the presence of an asparagine residue N186 in ThBgl2, which is replaced by the phenylalanine F180 in ThBgl1. This single amino acid substitution seems to be sufficient to create a polar environment that culminates with an increased availability of water molecules in ThBgl2 as compared to ThBgl1, thus conferring stronger hydrolyzing character to the former enzyme.

Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including β-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-β-1,3(4)-glucanase (EC 3.2.1.6), can cleave both β-1,3 and β-1,4 glycosidic bonds in glucans, such as laminarin, barley β-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-β-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of β-1,3 and β-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.

The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of β-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the "Newton's cradle" catalytic mechanism recently proposed for this subfamily.

Antibiotic resistance is a worldwide concern that requires a concerted action from physicians, patients, governmental agencies, and academia to prevent infections and the spread of resistance, track resistant bacteria, improve the use of current antibiotics, and develop new antibiotics. Despite the efforts spent so far, the current antibiotics in the market are restricted to only five general targets/pathways highlighting the need for basic research focusing on the discovery and evaluation of new potential targets. Here we interrogate two biosynthetic pathways as potentially druggable pathways in bacteria. The biosynthesis pathway for thiamine (vitamin B1), absent in humans, but found in many bacteria, including organisms in the group of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp.) and the biosynthesis pathway for pyridoxal 5'-phosphate and its vitamers (vitamin B6), found in S. aureus. Using current genomic data, we discuss the possibilities of inhibition of enzymes in the pathway and review the current state of the art in the scientific literature.