Heterogeneity of proteinases from the hyperthermophilic archaeobacterium, Pyrococcus furiosus.

In order to investigate the role of proteolysis in Pyrococcus furiosus, cultures were grown on a variety of complex and simple carbon substrates and assayed for proteolytic activity using azocasein as the protein substrate. Growth on complex protein substrates such as peptone or tryptone induced high levels of extracellular proteinase activity, whereas growth in the presence of the oligosaccharide maltose largely repressed proteinase activity. Gelatin SDS PAGE revealed thirteen proteolytically active bands in both cell extracts and culture supernatants, with apparent molecular weights ranging from 66kDa to 135kDa. It was concluded that these bands were not artefacts but probably discrete polypeptides. The band pattern was observed after a variety of treatments, which included different incubation times and temperature, SDS concentration, different proteolytic substrate (casein) and thiol-interchange mechanisms. Using a variety of chromatographic techniques, only the 66kDa proteinase could be resolved. Mono Q, Hydrogen Bond cellulose, sucrose density gradient centrifugation, and gel permeation under non-denaturing conditions all produced similar band patterns suggesting that the polypeptides existed as active aggregates or as a high molecular weight complex. The proteinase 'complex' from cell extracts was purified to near homogeneity with a yield of 35%. The molecular weight was estimated to be approximately 10⁶ Daltons. The pH optimum for activity was between 6 and 8, and the ''temperature optimum" was 100°C. At 95°C, the proteinase complex had a half-life of 69h. The proteinase complex was stable at 95°C in 6M urea, 10mM dithiothreitol and in 4.4M guanidinium chloride. Enhanced stability was observed in the presence of 0.1% and 1% Triton X-100, whereas the detergents SDS and CTAB produced a large destabilising effect. Organic solvents (ethanol, acetone and dimethylformamide) significantly reduced thermostability of the proteinase complex at 100°C. Enhanced stability of the complex was observed in the presence of 1M NaC1. Inhibition studies indicated that the activity of the complex was predominantly of a serine-type. Inhibition by EDTA of a PMSF-insensitive proteinase was observed in gelatin SDS gels, suggesting the presence of at least one metalloproteinase activity in the complex. A preliminary determination of substrate specificity indicated a preference for small basic amino acids and substrates containing phenylalanine adjacent to the leaving group

Structural characterization of the carbohydrate-binding module of NanA sialidase, a pneumococcal virulence factor

This research was supported by the University of St Andrews and grants provided by the Medical Research Council.Background: Streptococcus pneumoniae Neuraminidase A (NanA) is a multi-domain protein anchored to the bacterial surface. Upstream of the catalytic domain of NanA is a domain that conforms to the sialic acid-recognising CBM40 family of the CAZY (carbohydrate-active enzymes) database. This domain has been identified to play a critical role in allowing the bacterium to promote adhesion and invasion of human brain microvascular endothelial cells, and hence may play a key role in promoting bacterial meningitis. In addition, the CBM40 domain has also been reported to activate host chemokines and neutrophil recruitment during infection. Results: Crystal structures of both apo- and holo- forms of the NanA CBM40 domain (residues 121 to 305), have been determined to 1.8 angstrom resolution. The domain shares the fold of other CBM40 domains that are associated with sialidases. When in complex with alpha 2,3- or alpha 2,6-sialyllactose, the domain is shown to interact only with the terminal sialic acid. Significantly, a deep acidic pocket adjacent to the sialic acid-binding site is identified, which is occupied by a lysine from a symmetry-related molecule in the crystal. This pocket is adjacent to a region that is predicted to be involved in protein-protein interactions. Conclusions: The structural data provide the details of linkage-independent sialyllactose binding by NanA CBM40 and reveal striking surface features that may hold the key to recognition of binding partners on the host cell surface. The structure also suggests that small molecules or sialic acid analogues could be developed to fill the acidic pocket and hence provide a new therapeutic avenue against meningitis caused by S. pneumoniae.Publisher PDFPeer reviewe

The impact of viral infection on the chemistries of the Earth’s most abundant photosynthesisers : metabolically talented aquatic cyanobacteria

Funding: The authors thank BBSRC BB/T017058/1 (YW) IBioIC, MASTS, Xanthella (SF) and the Royal Society (RJMG) for financial support.Cyanobacteria are the most abundant photosynthesizers on earth, and as such, they play a central role in marine metabolite generation, ocean nutrient cycling, and the control of planetary oxygen generation. Cyanobacteriophage infection exerts control on all of these critical processes of the planet, with the phage-ported homologs of genes linked to photosynthesis, catabolism, and secondary metabolism (marine metabolite generation). Here, we analyze the 153 fully sequenced cyanophages from the National Center for Biotechnology Information (NCBI) database and the 45 auxiliary metabolic genes (AMGs) that they deliver into their hosts. Most of these AMGs are homologs of those found within cyanobacteria and play a key role in cyanobacterial metabolism-encoding proteins involved in photosynthesis, central carbon metabolism, phosphate metabolism, methylation, and cellular regulation. A greater understanding of cyanobacteriophage infection will pave the way to a better understanding of carbon fixation and nutrient cycling, as well as provide new tools for synthetic biology and alternative approaches for the use of cyanobacteria in biotechnology and sustainable manufacturing.Publisher PDFPeer reviewe

Halogenases:a palette of emerging opportunities for synthetic biology–synthetic chemistry and C–H functionalisation

Authors thank ERC GenoChemetics (FP7/2007-2013/ERC consolidator grant GCGXC grant agreement no. 614779 RJMG) for funding, BBSRC Follow on Funding, Synthetic Biology Highlight award. RSE Enterprise Fellowship (SVS), China Scholarship Council and EPSRC CRITICAT EP/L016419/1 for studentship support (Y. Z. & S. M. respectively).The enzymatic generation of carbon–halogen bonds is a powerful strategy used by both nature and synthetic chemists to tune the bioactivity, bioavailability and reactivity of compounds, opening up the opportunity for selective C–H functionalisation. Genes encoding halogenase enzymes have recently been shown to transcend all kingdoms of life. These enzymes install halogen atoms into aromatic and less activated aliphatic substrates, achieving selectivities that are often challenging to accomplish using synthetic methodologies. Significant advances in both halogenase discovery and engineering have provided a toolbox of enzymes, enabling the ready use of these catalysts in biotransformations, synthetic biology, and in combination with chemical catalysis to enable late stage C–H functionalisation. With a focus on substrate scope, this review outlines the mechanisms employed by the major classes of halogenases, while in parallel, it highlights key advances in the utilisation of the combination of enzymatic halogenation and chemical catalysis for C–H activation and diversification.Publisher PDFPeer reviewe

Neuraminidase-deficient Sendai virus HN mutants provide protection from homologous superinfection

Binding of hemagglutinin-neuraminidase proteins (HN) to sialylated receptors initiates the infection process of several paramyxoviruses, whereas later in the viral life cycle, the neuramindase (NA) activity of newly synthesized HN destroys all receptors. Prior to NA action, expressed HN has to bind the receptor. To evaluate this HN–receptor complex with respect to receptor inactivation, three temperature-sensitive Sendai virus HN mutants carrying amino acid exchanges at positions 262, 264 and/or 461 were created that uncoupled NA activity from receptor binding at 39°C. Interestingly, at elevated temperature, when there is no detectable neuramindase activity, all infected cells are protected against homologous superinfection. Mutated HN protein on the cell surface is mainly bound to sialylated cell-surface components but can be released by treatment with NA. Thus, continuous binding to HN already inactivates the receptors quantitatively. Furthermore, mutant HN bound to receptors is prevented from being incorporated into virus particles in the absence of NA. It is shown here for the first time that during paramyxoviral infection, quantitative receptor inactivation already occurs due to binding of receptors to expressed HN protein without involvement of NA and is independent of NA activity of viral progeny. NA subsequently functions in the release of HN from the complex, coupled with desialysation of receptors. These findings could have implications for further antiviral drug development

Newcastle Disease Virus in Madagascar: Identification of an Original Genotype Possibly Deriving from a Died Out Ancestor of Genotype IV

In Madagascar, Newcastle disease (ND) has become enzootic after the first documented epizootics in 1946, with recurrent annual outbreaks causing mortality up to 40%. Four ND viruses recently isolated in Madagascar were genotypically and pathotypically characterised. By phylogenetic inference based on the F and HN genes, and also full-genome sequence analyses, the NDV Malagasy isolates form a cluster distant enough to constitute a new genotype hereby proposed as genotype XI. This new genotype is presumably deriving from an ancestor close to genotype IV introduced in the island probably more than 50 years ago. Our data show also that all the previously described neutralising epitopes are conserved between Malagasy and vaccine strains. However, the potential implication in vaccination failures of specific amino acid substitutions predominantly found on surface-exposed epitopes of F and HN proteins is discussed

A study of the protozoan Tritrichomonas foetus with particular relevance to glycosidase production

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