Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other α/β-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.

Expression of Distal-less, dachshund, and optomotor blind in Neanthes arenaceodentata (Annelida, Nereididae) does not support homology of appendage-forming mechanisms across the Bilateria

The similarity in the genetic regulation of arthropod and vertebrate appendage formation has been interpreted as the product of a plesiomorphic gene network that was primitively involved in bilaterian appendage development and co-opted to build appendages (in modern phyla) that are not historically related as structures. Data from lophotrochozoans are needed to clarify the pervasiveness of plesiomorphic appendage forming mechanisms. We assayed the expression of three arthropod and vertebrate limb gene orthologs, Distal-less (Dll), dachshund (dac), and optomotor blind (omb), in direct-developing juveniles of the polychaete Neanthes arenaceodentata. Parapodial Dll expression marks premorphogenetic notopodia and neuropodia, becoming restricted to the bases of notopodial cirri and to ventral portions of neuropodia. In outgrowing cephalic appendages, Dll activity is primarily restricted to proximal domains. Dll expression is also prominent in the brain. dac expression occurs in the brain, nerve cord ganglia, a pair of pharyngeal ganglia, presumed interneurons linking a pair of segmental nerves, and in newly differentiating mesoderm. Domains of omb expression include the brain, nerve cord ganglia, one pair of anterior cirri, presumed precursors of dorsal musculature, and the same pharyngeal ganglia and presumed interneurons that express dac. Contrary to their roles in outgrowing arthropod and vertebrate appendages, Dll, dac, and omb lack comparable expression in Neanthes appendages, implying independent evolution of annelid appendage development. We infer that parapodia and arthropodia are not structurally or mechanistically homologous (but their primordia might be), that Dll’s ancestral bilaterian function was in sensory and central nervous system differentiation, and that locomotory appendages possibly evolved from sensory outgrowths

Biostratigraphic Evidence Relating to the Age-Old Question of Hannibal's Invasion of Italy, II: Chemical Biomarkers and Microbial Signatures

Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesAs discussed in Part I, a large accumulation of mammalian faeces at the mire site in the upper Guil Valley near Mt. Viso, dated to 2168 cal 14C yr., provides the first evidence of the passage of substantial but indeterminate numbers of mammals within the time frame of the Punic invasion of Italia. Specialized organic biomarkers bound up in a highly convoluted and bioturbated bed constitute an unusual anomaly in a histosol comprised of fibric and hemist horizons that are usually expected to display horizontal bedding. The presence of deoxycholic acid and ethylcoprostanol derived from faecal matter, coupled with high relative numbers of Clostridia 16S rRNA genes, suggests a substantial accumulation of mammalian faeces at the site over 2000 years ago. The results reported here constitute the first chemical and biological evidence of the passage of large numbers of mammals, possibly indicating the route of the Hannibalic army at this time. Combined with the geological analysis reported in Part I, these data provide a background supporting the need for further historical archaeological exploration in this area.Ye

The extracellular juncture domains in the intimin passenger adopt a constitutively extended conformation inducing restraints to its sphere of action

Enterohemorrhagic and enteropathogenic Escherichia coli are among the most important food-borne pathogens, posing a global health threat of bloody diarrhea and hemolytic uremic syndrome outbreaks. The virulence factor intimin is essential for attachment and internalization into the host cells of a broad range of effacing pathogens. Intimin is translocated to the bacterial outer membrane and includes an extracellular passenger, which consists of four bacterial immunoglobulin (Big) like domains, D00-D2, extending into the fifth subdomain, a C-terminal lectin domain (D3) that binds to the Tir-receptor on the host cell. Here, we present the crystal structures of D00-D0 at 1.5 Å and D0-D1 at 1.8 Å resolution that confirm that the passenger of intimin has five distinct domains. SAXS and MD simulations show that the connector region between D00-D0 has a higher degree of rigidity and D00 likely functions as a juncture domain at the outer membrane-extracellular medium interface. We describe D00 as a Big domain with a specific topology found in the equivalent position in a broad range of other inverse autotransporters, including the structurally uncharacterized D0 domain in invasin. The accumulated data allows us to model the complete passenger of intimin and propose functionality to the Big domains, D00-D0-D1, extending directly from the membrane

Biostratigraphic Evidence Relating to the Age-Old Question of Hannibal's Invasion of Italy, I: History and Geological Reconstruction

Controversy over the alpine route that Hannibal of Carthage followed from the Rhône Basin into Italia has raged amongst classicists and ancient historians for over two millennia. The motivation for identifying the route taken by the Punic Army through the Alps lies in its potential for identifying sites of historical archaeological significance and for the resolution of one of history's most enduring quandaries. Here, we present stratigraphic, geochemical and microbiological evidence recovered from an alluvial floodplain mire located below the Col de la Traversette (~3000 m asl—above sea level) on the French/Italian border that potentially identifies the invasion route as the one originally proposed by Sir Gavin de Beer (de Beer 1974). The dated layer is termed the MAD bed (mass animal deposition) based on disrupted bedding, greatly increased organic carbon and key/specialized biological components/compounds, the latter reported in Part II of this paper. We propose that the highly abnormal churned up (bioturbated) bed was contaminated by the passage of Hannibal's animals, possibly thousands, feeding and watering at the site, during the early stage of Hannibal's invasion of Italia (218 bc)

THE ASSOCIATION OF DEPRESSION WITH C-REACTIVE PROTEIN (THE DATA OF ESSE-RF EPIDEMIOLOGICAL STUDY)

Aim. To study the association of depression with a high-sensitivity C-reactive protein (hsCRP) level, taking into account the main risk factors and noncommunicable diseases in Russia residents.Material and methods. The data of ESSE-RF multicenter study (a representative sample of the unorganized male and female population aged 25-64 years from 8 regions surveyed in 2012-2014) were used in the work. A total 11884 people were involved into the study including 35.9% men. The examination included a survey on the standard questionnaire containing data on disease history, etc. The level of depression was assessed by the validated in Russian Hospital Anxiety and Depression Scale (HADS, 1983). hsCRP level was determined in all patients.Results. The continuing association between elevated levels of depression (HADS-D ≥8+) and high level of hsCRP ≥3.0 mg/l (odds ratio [OR] 1.15; 95% confidence interval [CI] 1.03-1.27; p=0.009) was found in the multivariate model, after adjustment for sex, age, education, and risk factors. Reducing of the relationship of elevated levels of depression with a high level of hsCRP (OR 1.11; 95% CI 1.00-1.24; p=0.048) was found with the additional introduction of diseases in the model. This relationship was reduced to not statistically significant level (OR 1.08; 95% CI 0.98-1.20; p=0.134) in the full model adjusted for regions.Conclusion. The reduced association of depression with hsCRP ajusted for aggregate risk factors was found in the study. This suggests about multifactor affecting on this relationship

Coordinated spatial and temporal expression of Hox genes during embryogenesis in the acoel Convolutriloba longifissura

Background: Hox genes are critical for patterning the bilaterian anterior-posterior axis. The evolution of their clustered genomic arrangement and ancestral function has been debated since their discovery. As acoels appear to represent the sister group to the remaining Bilateria (Nephrozoa), investigating Hox gene expression will provide an insight into the ancestral features of the Hox genes in metazoan evolution. Results: We describe the expression of anterior, central and posterior class Hox genes and the ParaHox ortholog Cdx in the acoel Convolutriloba longifissura. Expression of all three Hox genes begins contemporaneously after gastrulation and then resolves into staggered domains along the anterior-posterior axis, suggesting that the spatial coordination of Hox gene expression was present in the bilaterian ancestor. After early surface ectodermal expression, the anterior and central class genes are expressed in small domains of putative neural precursor cells co-expressing ClSoxB1, suggesting an evolutionary early function of Hox genes in patterning parts of the nervous system. In contrast, the expression of the posterior Hox gene is found in all three germ layers in a much broader posterior region of the embryo. Conclusion: Our results suggest that the ancestral set of Hox genes was involved in the anteriorposterior patterning of the nervous system of the last common bilaterian ancestor and were later co-opted for patterning in diverse tissues in the bilaterian radiation. The lack of temporal colinearity of Hox expression in acoels may be due to a loss of genomic clustering in this clade or, alternatively, temporal colinearity may have arisen in conjunction with the expansion of the Hox cluster in the Nephrozoa

Isolation and Characterization of EstC, a New Cold-Active Esterase from Streptomyces coelicolor A3(2)

The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5–9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6–11. The enzyme was active toward short-chain p-nitrophenyl esters (C2–C12), displaying optimal activity with the valerate (C5) ester (kcat/Km = 737±77 s−1 mM−1). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors

The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology

Background: The major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest. Results: To explore early kinase evolution and regulation of Giardia biology, we cataloged the kinomes of three sequenced strains. Comparison with published kinomes and those of the excavates Trichomonas vaginalis and Leishmania major shows that Giardia's 80 core kinases constitute the smallest known core kinome of any eukaryote that can be grown in pure culture, reflecting both its early origin and secondary gene loss. Kinase losses in DNA repair, mitochondrial function, transcription, splicing, and stress response reflect this reduced genome, while the presence of other kinases helps define the kinome of the last common eukaryotic ancestor. Immunofluorescence analysis shows abundant phospho-staining in trophozoites, with phosphotyrosine abundant in the nuclei and phosphothreonine and phosphoserine in distinct cytoskeletal organelles. The Nek kinase family has been massively expanded, accounting for 198 of the 278 protein kinases in Giardia. Most Neks are catalytically inactive, have very divergent sequences and undergo extensive duplication and loss between strains. Many Neks are highly induced during development. We localized four catalytically active Neks to distinct parts of the cytoskeleton and one inactive Nek to the cytoplasm. Conclusions: The reduced kinome of Giardia sheds new light on early kinase evolution, and its highly divergent sequences add to the definition of individual kinase families as well as offering specific drug targets. Giardia's massive Nek expansion may reflect its distinctive lifestyle, biphasic life cycle and complex cytoskeleton