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

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

Pragmatic skills predict online counterfactual comprehension:Evidence from the N400

Counterfactual thought allows people to consider alternative worlds they know to be false. Communicating these thoughts through language poses a social-communicative challenge because listeners typically expect a speaker to produce true utterances, but counterfactuals per definition convey information that is false. Listeners must therefore incorporate overt linguistic cues (subjunctive mood, such as in If I loved you then) in a rapid way to infer the intended counterfactual meaning. The present EEG study focused on the comprehension of such counterfactual antecedents and investigated if pragmatic ability—the ability to apply knowledge of the social-communicative use of language in daily life—predicts the online generation of counterfactual worlds. This yielded two novel findings: (1) Words that are consistent with factual knowledge incur a semantic processing cost, as reflected in larger N400 amplitude, in counterfactual antecedents compared to hypothetical antecedents (If sweets were/are made of sugar). We take this to suggest that counterfactuality is quickly incorporated during language comprehension and reduces online expectations based on factual knowledge. (2) Individual scores on the Autism Quotient Communication subscale modulated this effect, suggesting that individuals who are better at understanding the communicative intentions of other people are more likely to reduce knowledge-based expectations in counterfactuals. These results are the first demonstration of the real-time pragmatic processes involved in creating possible worlds

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

Non-High Density Lipoprotein Cholesterol: A Modern Benchmark for Assessing Lipid Metabolism Disorders

Aim. To perform a population analysis of Non-High Density Lipoprotein Cholesterol level (non-HDL-c) in Russian population and to evaluate its association with cardiovascular events.Material and Methods. The material consisted of results obtained from 11 regions of the ESSE-RF1 Study and from 4 regions of the ESSE-RF2 Study. Study protocols were identical. The studies were performed in 2012-2014 and 2017, respectively. Endpoints were assessed in 19041 people aged 35-64 years. The median follow-up was 6.5 years in ESSE RF (1) and 3.8 years in ESSE RF(2). Analysis was performed for three lipid variables: total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and non-HDLC in two samples: the general population sample and the same sample without individuals with coronary heart disease (CHD), myocardial infarction (MI) and/or stroke history and not taking statins (the population sample of "without a history of cardiovascular diseases [CVD]". The analysis of nonlinear associations was performed using the generalized additive Cox model. The combined cardiovascular endpoint was represented by cardiovascular death and nonfatal MI and stroke. Traditional and laboratory FRs, socio-demographic parameters were analyzed. The significance level for all tested hypotheses was set to be 0.05.Results. The prevalence of elevated non-HDL-C level (>3.7 mmol/l) was found to be 74.6%. No gender differences were found: there was 74.6% for men and 74.5% for women. Both mean values and prevalence of elevated non-HDL-C were increased with age in women, and its level was slightly decreased in men after 55 years old. Almost all analyzed RFs were significantly associated with elevated non-HDL-C in these two population samples. In both samples elevated total CH and elevated LDL-C were associated with all-cause mortality after correction for all RFs. On the contrary, the non-HDL-C was associated with CVD combined end pints. It has been shown that the risk of these end points increases uniformly with increase in levels of non HDL cholesterol, no nonlinear associations were found.Conclusion. The results of a population-based analysis of non-HDL-C performed in the Russian population for the first time confirmed that elevated non-HDL-C levels contribute significantly to determining the risk of cardiovascular events in the medium term. It can be assumed that the new risk scales (SCORE2 and SCORE OP) proposed by the European Society of Cardiology and the European Society of Preventive Cardiology, which include non-HDL C instead of TC, will allow adequate assessment of 10-year cardiovascular risk for Russians. However, continued monitoring of endpoints in order to obtain stable associations is required