Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry

The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNAIle, the 2',3'-dialdehyde derivative of tRNAIle was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNAIle aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNAIleox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNAIle and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence 601KMSKS605. Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [32P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site

Study of the Interaction of Zinc Cation with Azithromycin and its Significance in the COVID-19 Treatment: A Molecular Approach

INTRODUCTION: On account of the current COVID-19 pandemic, we have explored the importance of azithromycin and zinc in the treatment of the coronavirus disease by studying the interaction between the cation Zn++ and azithromycin with the tools of the semi-empirical quantum mechanics PM3 method. METHODS: By this approach, the niche in which Zn++ is located was determined. Zn++ creates a strong clastic binding between an amine and a hydroxyl group located on the amino-hexose side-chain. Such an interaction serves as a shuttle and allows zinc cation to invade endocellular structures. RESULTS: In this triple collaborative association, the role of hydroxychloroquine would be more that of a chaotropic agent at plasmic membranes, which facilitates access to the azithromycin-Zn++ equipage into key internal compartments. CONCLUSION: Finally, we show that both azithromycin and Zn++ are susceptible to play a direct role against the replication and the assembly of SARS-CoV-2 particles

EF-Tu from the enacyloxin producing Frateuria W-315 strain: Structure/activity relationship and antibiotic resistance

International audienceIn this report, we have demonstrated that the poly(U)-dependent poly(Phe) synthesis activity of elon-gator factor Tu (EF-Tu) from the enacyloxin producing strain Frateuria sp. W-315 is inhibited by the antibiotic similarly to that of Escherichia coli EF-Tu. The inhibitory effect of enacyloxin observed in a purified system was the same as that obtained with an S30 extract from E. coli or Frateuria sp. W-315, respectively, suggesting that antibiotic resistance of enacyloxin producing Frateuria sp. W-315 is not due neither to EF-Tu nor to other components of the translation machinery but to a still unknown mechanism. The EF-Tu gene, as PCR amplified from Frateuria W-315 genomic DNA and sequenced represented an ORF of 1191 nucleotides corresponding to 396 amino acids. This protein is larger than the product of tufA from E. coli by only two amino acid residues. Alignment of the amino acid sequence of EF-Tu from E. coli with those of Frateuria and Ralstonia solanacearum indicates on average 80% identical amino acid residues and 9.7% conservative replacements between EF-Tu Frateuria and EF-Tu E. coli, on one hand, and 97% identity and 1.7% conservative replacement between EF-Tu Frateuria and EF-Tu Ralstonia sol-anacearum, on the other hand. These strong primary structure similarities between EF-Tu from different origins are consistent with the fact that this factor is essential for the translation process in all kingdoms of life. Comparison of the effects of antibiotics on EF-Tu Frateuria and EF-Tu E. coli revealed that enacyloxin, kirromycin and pulvomycin exert a stronger stimulation of the GDP dissociation rate on EF-Tu Frateuria, while the effects of the antibiotics on the GDP association rate were comparable for the two EF-Tu species. Different mutants of EF-Tu E. coli were constructed with the help of site directed muta-genesis by changing one or several residues of EF-Tu E. coli by the corresponding residues of EF-Tu Frateuria. The single A45K substitution did not modify the intrinsic GTPase activity of EF-Tu E. coli. In contrast, a 2e3 fold stimulation of the intrinsic GTPase activity was observed with the single A42E, F46Y, Q48E and the double F46Y/Q48E substitution. Finally, up to a 7 fold stimulation was observed with the quadruple substitution (mutant A42E/A45K/F46Y/Q48E

Repositioning Adequate Antibiotics to Treat/Cure the Coronavirus Disease 2019 (COVID-19): Current Treatments and Future Directions

AIMS: Rational use of antibiotics against the betacoronavirus SARS-CoV-2 responsible for the COVID-19 pandemic. OBJECTIVE : Repositioning and repurposing adequate antibiotics to cure the Coronavirus Disease 2019 (COVID-19). BACKGROUND : It is widely accepted that viral infections such as the SARS-CoV-2 cannot be cured by antibiotics, whereas bacterial infections can. It is because the SARS-CoV-2 virus has no protein synthesis machinery (usually targeted by antibiotics) to produce from its RNA genome, the viral proteins and enzymes essential for its replication and/or for the assembly of viral particles. However, the antibiotics must be capable of inhibiting the ribosomes of the protein synthesis machinery of the SARS-CoV-2-infected human host cells, in order to prevent them from synthesizing new proteins that they do not need, but are needed for the virus to spread. Unfortunately, the only antibiotic capable of selectively inhibiting the human 80S ribosomes, namely cycloheximide, was found to be a poisonous drug for the mammals. Therefore, the only possibility is to search for the antibiotics that are capable of inhibiting both bacterial and eukaryal ribosomes, in order to prevent at the same time the ribosomes of the infected human host cells from synthesizing the proteins and enzymes for the SARS-CoV-2 virus, and those of the eventual opportunistic pathogenic bacteria from developing pneumonia. METHODS: First, we have used a molecular modeling study involving the tools of the semi-empirical quantum mechanics PM3 method to study the interaction between the cation Zn++ and all the molecules considered as zinc transporters in this report. By this approach, the niche in which Zn++ is located was determined. Such an interaction serves as a shuttle and allows zinc cation to invade endocellular structures in the SARS-CoV-2-infected human host cells. Second, we have measured the poly (U)-dependent poly (Phe) synthesis activity of human 80S ribosomes in the presence of increasing concentrations of four antibiotics of the class of the macrolides, namely erythromycin, azithromycin, clarithromycin and telithromycin. This experiment led us to determine for each macrolide, the half-inhibitory concentration (IC50) that is the concentration of antibiotic corresponding to 50% inhibition of the activity of the human 80S ribosomes. Finally, we have analyzed previously published data from the group of Nierhaus (Berlin) on the competition between the incoming aminoacyl-tRNA and the antibiotic tetracycline for the binding to the ribosomal A-site on the E. coli 70S or rabbit liver 80S ribosomes. This led to the conclusion by the authors that tetracycline most likely binds to corresponding sites in 70S and 80S ribosomes with comparable affinity. [...

Enzyme-induced covalent modification of methionyl-tRNA synthetase from Bacillus stearothermophilus by methionyl-adenylate: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.

International audienceMethionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the epsilon-NH2 group of lysyl residues. The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 +/- 0.1 and 4.3 +/- 0.1 mol of [14C]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetase, respectively. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues

Covalent methionylation of Escherichia coli methionyl-tRNA synthetase: Identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry

International audienceMethionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by methionyl-tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the epsilon-NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl-adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a monomeric truncated methionyl-tRNA synthetase species, the enzymic activities of [P-32]PP1-ATP isotopic exchange and tRNA(Met) aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNA(Met) protected the enzyme against inactivation and methionine incorporation. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-114, -132, -142 (or -147), -270, -282. -335, -362, -402, -439, -465, and -547 of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114-150], [270-362], and [402-465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA