Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes

Hen egg-white lysozyme has long been the paradigm for enzymatic glycosyl hydrolysis with retention of configuration, with a protonated carboxylic acid and a deprotonated carboxylate participating in general acid-base catalysis. In marked contrast, the retaining chitin degrading enzymes from glycosyl hydrolase families 18 and 20 all have a single glutamic acid as the catalytic acid but lack a nucleophile on the enzyme. Both families have a catalytic (βα)8-barrel domain in common. X-ray structures of three different chitinolytic enzymes complexed with substrates or inhibitors identify a retaining mechanism involving a protein acid and the carbonyl oxygen atom of the substrate’s C2 N-acetyl group as the nucleophile. These studies unambiguously demonstrate the distortion of the sugar ring toward a sofa conformation, long postulated as being close to that of the transition state in glycosyl hydrolysis.

Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages

This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments

Crystal structure of the RNA polymerase domain of the West Nile Virus non-structural protein 5

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue ( DENV) and West Nile ( WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase ( RdRp) activity of the non-structural protein 5 ( NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-angstrom resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNVRdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase ( MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain ( modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role

Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde

The Ruegeria pomeroyi acuI Gene Has a Role in DMSP Catabolism and Resembles yhdH of E. coli and Other Bacteria in Conferring Resistance to Acrylate

The Escherichia coli YhdH polypeptide is in the MDR012 sub-group of medium chain reductase/dehydrogenases, but its biological function was unknown and no phenotypes of YhdH− mutants had been described. We found that an E. coli strain with an insertional mutation in yhdH was hyper-sensitive to inhibitory effects of acrylate, and, to a lesser extent, to those of 3-hydroxypropionate. Close homologues of YhdH occur in many Bacterial taxa and at least two animals. The acrylate sensitivity of YhdH− mutants was corrected by the corresponding, cloned homologues from several bacteria. One such homologue is acuI, which has a role in acrylate degradation in marine bacteria that catabolise dimethylsulfoniopropionate (DMSP) an abundant anti-stress compound made by marine phytoplankton. The acuI genes of such bacteria are often linked to ddd genes that encode enzymes that cleave DMSP into acrylate plus dimethyl sulfide (DMS), even though these are in different polypeptide families, in unrelated bacteria. Furthermore, most strains of Roseobacters, a clade of abundant marine bacteria, cleave DMSP into acrylate plus DMS, and can also demethylate it, using DMSP demethylase. In most Roseobacters, the corresponding gene, dmdA, lies immediately upstream of acuI and in the model Roseobacter strain Ruegeria pomeroyi DSS-3, dmdA-acuI were co-regulated in response to the co-inducer, acrylate. These observations, together with findings by others that AcuI has acryloyl-CoA reductase activity, lead us to suggest that YdhH/AcuI enzymes protect cells against damaging effects of intracellular acryloyl-CoA, formed endogenously, and/or via catabolising exogenous acrylate. To provide “added protection” for bacteria that form acrylate from DMSP, acuI was recruited into clusters of genes involved in this conversion and, in the case of acuI and dmdA in the Roseobacters, their co-expression may underpin an interaction between the two routes of DMSP catabolism, whereby the acrylate product of DMSP lyases is a co-inducer for the demethylation pathway

The Mycobacterium tuberculosis Drugome and Its Polypharmacological Implications

We report a computational approach that integrates structural bioinformatics, molecular modelling and systems biology to construct a drug-target network on a structural proteome-wide scale. The approach has been applied to the genome of Mycobacterium tuberculosis (M.tb), the causative agent of one of today's most widely spread infectious diseases. The resulting drug-target interaction network for all structurally characterized approved drugs bound to putative M.tb receptors, we refer to as the ‘TB-drugome’. The TB-drugome reveals that approximately one-third of the drugs examined have the potential to be repositioned to treat tuberculosis and that many currently unexploited M.tb receptors may be chemically druggable and could serve as novel anti-tubercular targets. Furthermore, a detailed analysis of the TB-drugome has shed new light on the controversial issues surrounding drug-target networks [1]–[3]. Indeed, our results support the idea that drug-target networks are inherently modular, and further that any observed randomness is mainly caused by biased target coverage. The TB-drugome (http://funsite.sdsc.edu/drugome/TB) has the potential to be a valuable resource in the development of safe and efficient anti-tubercular drugs. More generally the methodology may be applied to other pathogens of interest with results improving as more of their structural proteomes are determined through the continued efforts of structural biology/genomics

Plasticity of the β-Trefoil Protein Fold in the Recognition and Control of Invertebrate Predators and Parasites by a Fungal Defence System

Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity

Effects of school-based interventions on mental health stigmatization: a systematic review

Stigmatizing, or discriminatory, perspectives and behaviour, which target individuals on the basis of their mental health, are observed in even the youngest school children. We conducted a systematic review of the published and unpublished, scientific literature concerning the benefits and harms of school-based interventions, which were directed at students 18 years of age or younger to prevent or eliminate such stigmatization. Forty relevant studies were identified, yet only a qualitative synthesis was deemed appropriate. Five limitations within the evidence base constituted barriers to drawing conclusive inferences about the effectiveness and harms of school-based interventions: poor reporting quality, a dearth of randomized controlled trial evidence, poor methods quality for all research designs, considerable clinical heterogeneity, and inconsistent or null results. Nevertheless, certain suggestive evidence derived both from within and beyond our evidence base has allowed us to recommend the development, implementation and evaluation of a curriculum, which fosters the development of empathy and, in turn, an orientation toward social inclusion and inclusiveness. These effects may be achieved largely by bringing especially but not exclusively the youngest children into direct, structured contact with an infant, and likely only the oldest children and youth into direct contact with individuals experiencing mental health difficulties. The possible value of using educational activities, materials and contents to enhance hypothesized benefits accruing to direct contact also requires investigation. Overall, the curriculum might serve as primary prevention for some students and as secondary prevention for others

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-ef-fective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans—a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxida-tive cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications