Sébastien P. Blais

A Weak Fe–O Bond in the Oxygenated Complex of the Nitric-oxide Synthase of Staphylococcus aureus

Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixing reduced saNOS with oxygen and decayed to the ferric enzyme with kinetics that were dependent on the substrate l-arginine and the cofactor H4B. The oxygenated complex displayed a Soret absorption band centered at 430 nm. Resonance Raman spectroscopy revealed that it can be described as a ferric superoxide form (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Fe}^{\mathrm{III}}\mathrm{O}_{2}^{-}\) \end{document}) with a single νO–O mode at 1135 cm–1. In the presence of l-arginine, an additional νO–O mode at 1123 cm–1 was observed, indicating an increased π back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the νFe–O mode of a NOS has been observed. The low frequency of this mode, at 517 cm–1, points to an oxygenated complex that differs from that of P450cam. The electronic structure of the oxygenated complex and the effect of l-arginine are discussed in relation to the kinetic properties of saNOS and other NOS.

Plasmodial Aspartyl-tRNA Synthetases and Peculiarities in Plasmodium falciparum

Distinctive features of aspartyl-transfer RNA (tRNA) synthetases (AspRS) from the protozoan Plasmodium genus are described. These apicomplexan AspRSs contain 29–31 amino acid insertions in their anticodon binding domains, a remarkably long N-terminal appendix that varies in size from 110 to 165 amino acids and two potential initiation codons. This article focuses on the atypical functional and structural properties of Plasmodium falciparum cytosolic AspRS, the causative parasite of human malaria. This species encodes a 626 or 577 amino acids AspRS depending on whether initiation starts on the first or second in-frame initiation codon. The longer protein has poor solubility and a propensity to aggregate. Production of the short version was favored as shown by the comparison of the recombinant protein with endogenous AspRS. Comparison of the tRNA aminoacylation activity of wild-type and mutant parasite AspRSs with those of yeast and human AspRSs revealed unique properties. The N-terminal extension contains a motif that provides unexpectedly strong RNA binding to plasmodial AspRS. Furthermore, experiments demonstrated the requirement of the plasmodial insertion for AspRS dimerization and, therefore, tRNA aminoacylation and other putative functions. Implications for the parasite biology are proposed. These data provide a robust background for unraveling the precise functional properties of the parasite AspRS and for developing novel lead compounds against malaria, targeting its idiosyncratic domains.

Inhibition of Helicobacter pylori aminoacyl-tRNA amidotransferase by chloramphenicol analogs

Genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthetase in many bacteria and all known archaea. In these microorganisms, glutaminyl-tRNA(Gln) (Gln-tRNA(Gln)) and/or asparaginyl-tRNA(Asn) (Asn-tRNA(Asn)) are synthesized via an indirect pathway involving side chain amidation of misacylated glutamyl-tRNA(Gln) (Glu-tRNA(Gln)) and/or aspartyl-tRNA(Asn) (Asp-tRNA(Asn)) by an amidotransferase. A series of chloramphenicol analogs have been synthesized and evaluated as inhibitors of Helicobacter pylori GatCAB amidotransferase. Compound 7a was identified as the most active competitive inhibitor of the transamidase activity with respect to Asp-tRNA(Asn) (K(m)=2μM), with a K(i) value of 27μM.

Correction: tRNAGlu Increases the Affinity of Glutamyl-tRNA Synthetase for Its Inhibitor Glutamyl-Sulfamoyl-Adenosine, an Analogue of the Aminoacylation Reaction Intermediate Glutamyl-AMP: Mechanistic and Evolutionary Implications.

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of –TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant K d is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.

Cost-effectiveness of FreeO2 in patients with chronic obstructive pulmonary disease hospitalised for acute exacerbations: analysis of a pilot study in Quebec

Objective Conduct a cost-effectiveness analysis of FreeO2 technology versus manual oxygen-titration technology for patients with chronic obstructive pulmonary disease (COPD) hospitalised for acute exacerbations. Setting Tertiary acute care hospital in Quebec, Canada. Participants 47 patients with COPD hospitalised for acute exacerbations. Intervention An automated oxygen-titration and oxygen-weaning technology. Methods and outcomes The costs for hospitalisation and follow-up for 180 days were calculated using a microcosting approach and included the cost of FreeO2 technology. Incremental cost-effectiveness ratios (ICERs) were calculated using bootstrap resampling with 5000 replications. The main effect variable was the percentage of time spent at the target oxygen saturation (SpO2). The other two effect variables were the time spent in hyperoxia (target SpO2+5%) and in severe hypoxaemia (SpO2 <85%). The resamplings were based on data from a randomised controlled trial with 47 patients with COPD hospitalised for acute exacerbations. Results FreeO2 generated savings of 20.7% of the per-patient costs at 180 days (ie, −

Cardiac Implantable Electronic Device Infection: Detailed Analysis of Cost Implications

C2959.71). This decrease is nevertheless not significant at the 95% threshold (P=0.13), but the effect variables all improved (P<0.001). The improvement in the time spent at the target SpO2 was 56.3%. The ICERs indicate that FreeO2 technology is more cost-effective than manual oxygen titration with a savings of −

Inhibition of Helicobacter pylori Glu‐tRNAGln amidotransferase by novel analogues of the putative transamidation intermediate

C96.91 per percentage point of time spent at the target SpO2 (95% CI −301.26 to 116.96). Conclusion FreeO2 technology could significantly enhance the efficiency of the health system by reducing per-patient costs at 180 days. A study with a larger patient sample needs to be carried out to confirm these preliminary results. Trial registration number NCT01393015; Post-results.

Mechanism of a GatCAB Amidotransferase: Aspartyl-tRNA Synthetase Increases Its Affinity for Asp-tRNAAsn and Novel Aminoacyl-tRNA Analogues Are Competitive Inhibitors†

BACKGROUND Infections of cardiac implantable electronic devices (CIED) are associated with significant morbidity and mortality. Despite many preventive measures, this condition is associated with significant costs for the health care system. METHODS We retrospectively analyzed all infection cases referred for lead extraction at a single university hospital over 1 year (2015-2016). We then calculated all costs related to the infection episode per patient using hospital databases and charts review. RESULTS Thirty-eight patients with CIED infections (29% women-mean age 71 ± 14 years) were referred for lead extraction (27 pocket infections, 11 endocarditis). Devices were mainly pacemakers (60%). When the pathogen was identified, Staphylococcus aureus methicillin sensitive was the main cause. Extraction was performed in all but 3 cases (92%). One death occurred in the nonextracted group. Respective durations of hospitalization and intravenous and antibiotic administration for patients undergoing extraction were 21 and 36 days. The calculated mean total cost for CIED infection management was CAD

Synthesis of β-ketophosphonate analogs of glutamyl and glutaminyl adenylate, and selective inhibition of the corresponding bacterial aminoacyl-tRNA synthetases

29,907 (median: 26,879; range: CAD

A C-Truncated Glutamyl-tRNA Synthetase Specific for tRNAGlu Is Stimulated by Its Free Complementary Distal Domain: Mechanistic and Evolutionary Implications

4,827-