glutaminyl-tRNA Synthetase Research Articles

Evolution and variation in amide aminoacyl-tRNA synthesis.

The amide proteogenic amino acids, asparagine and glutamine, are two of the twenty amino acids used in translation by all known life. The aminoacyl-tRNA synthetases for asparagine and glutamine, asparaginyl-tRNA synthetase and glutaminyl tRNA synthetase, evolved after the split in the last universal common ancestor of modern organisms. Before that split, life used two-step indirect pathways to synthesize asparagine and glutamine on their cognate tRNAs to form the aminoacyl-tRNA used in translation. These two-step pathways were retained throughout much of the bacterial and archaeal domains of life and eukaryotic organelles. The indirect routes use non-discriminating aminoacyl-tRNA synthetases (non-discriminating aspartyl-tRNA synthetase and non-discriminating glutamyl-tRNA synthetase) to misaminoacylate the tRNA. The misaminoacylated tRNA formed is then transamidated into the amide aminoacyl-tRNA used in protein synthesis by tRNA-dependent amidotransferases (GatCAB and GatDE). The enzymes and tRNAs involved assemble into complexes known as transamidosomes to help maintain translational fidelity. These pathways have evolved to meet the varied cellular needs across a diverse set of organisms, leading to significant variation. In certain bacteria, the indirect pathways may provide a means to adapt to cellular stress by reducing the fidelity of protein synthesis. The retention of these indirect pathways versus acquisition of asparaginyl-tRNA synthetase and glutaminyl tRNA synthetase in lineages likely involves a complex interplay of the competing uses of glutamine and asparagine beyond translation, energetic costs, co-evolution between enzymes and tRNA, and involvement in stress response that await further investigation.

Allosteric inhibition of tRNA synthetase Gln4 by N-pyrimidinyl-β-thiophenylacrylamides exerts highly selective antifungal activity

Candida species are among the most prevalent causes of systemic fungal infections, which account for ∼1.5 million annual fatalities. Here, we build on a compound screen that identified the molecule N-pyrimidinyl-β-thiophenylacrylamide (NP-BTA), which strongly inhibits Candida albicans growth. NP-BTA was hypothesized to target C.albicans glutaminyl-tRNA synthetase, Gln4. Here, we confirmed through invitro amino-acylation assays NP-BTA is a potent inhibitor of Gln4, and we defined how NP-BTA arrests Gln4's transferase activity using co-crystallography. This analysis also uncovered Met496 as a critical residue for the compound's species-selective target engagement and potency. Structure-activity relationship (SAR) studies demonstrated the NP-BTA scaffold is subject to oxidative and non-oxidative metabolism, making it unsuitable for systemic administration. In a mouse dermatomycosis model, however, topical application of the compound provided significant therapeutic benefit. This work expands the repertoire of antifungal protein synthesis target mechanisms and provides a path to develop Gln4 inhibitors.

Structural analysis of the YqeY proteins from Campylobacter jejuni and Vibrio parahaemolyticus

YqeY is a functionally and structurally uncharacterized protein that is ubiquitously expressed in bacteria. To gain structural insights into the function of YqeY, we determined the crystal structures of the Campylobacter jejuni and Vibrio parahaemolyticus YqeY proteins (cjYqeY and vpYqeY, respectively) and analyzed the structural and functional roles of conserved residues via a mutational study. Both cjYqeY and vpYqeY were found to adopt a two-domain structure consisting of an N-terminal four-α-helix domain and a C-terminal three-α-helix domain, with a relatively flexible interdomain orientation. The YqeY structure is unique in its linkage of the two α-helix domains although the C-terminal YqeY domain is structurally homologous to the terminal appendages of glutaminyl-tRNA synthetase and tRNA-dependent amidotransferase. We identified six conserved YqeY residues (Y67, R72, E82, Y89, P91, and G119) and evaluated their roles in protein stability via alanine mutation using a thermal shift assay. Residues Y67, R72, Y89, and P91 were shown to be required to maintain the structural integrity of YqeY. In contrast, residues E82 and G119 were not found to be essential for protein stability and are highly likely to contribute to the biological function of YqeY.

Complexation of some d-metals with N-benzyl-N-nitrosohydroxylamine derivatives. Crystal and molecular structure of diaquabis[N-benzyl-N-nitrosohydroxylaminato-κ2O,O′]cobaltl(II) and in silico target fishing

N-nitrosohydroxylamine derivatives are dubbed as chelating agents. The complexation of bivalent metals with N-benzyl-N-nitrosohydroxylamine (L1) and N-(2F-benzyl)-N-nitrosohydroxylamine (L2) were studied with DFT B3LYP modeling, electronic spectroscopy, and X-Ray diffraction. The 3d-metal cations showed good affinity to N-nitroso-N-hydroxylamine derivatives and formed complex compounds with high degree of covalence. Their absorption bands shifted towards the spectra of neutral forms of the non-coordinated ligands. The value of the shift increased in a series of Mn2+ < Co2+ < Ni2+ < Cu2+ > Zn2+ > Cd2+. The determined formation constants of the complexes lied in the range of 9.86 – 11.58. The crystal structure of Co(L1)2(H2O)2 showed that the organic species act as bidental chelating ligands occupying cis-positions in the coordination sphere of Co(II) cation. In the lattice, the molecules of the compound formed parallel layers which arranged in columns stabilized using a set of intermolecular hydrogen bonds. These ligands obeyed Lipinski’s and Veber’s rules of drug discovery pipeline. Besides, L1 and L2 also had half-life values of 0.7and 0.4 h, oral bioavailability score of 0.55, and positive blood–brain barrier permeability indicating their potential as therapeutic lead-like compounds. Further, potential targets were screened out computationally. L1 predominantly interacted with family A of G-protein coupled receptors and enzymes while L2 also interacted with protease and nuclear receptors in addition to the targets of its parent molecule, L1. Essentially, glutaminyl-tRNA synthetase and lumazine synthase were suggested as top-scored targets. In sum up, these ligands may be antimicrobial lead-like molecules suitable to be submitted to the in vitro validation.

Circadian clock control of tRNA synthetases in Neurospora crassa.

Background: In Neurospora crassa, the circadian clock controls rhythmic mRNA translation initiation through regulation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2). Active CPC-3 phosphorylates and inactivates eIF2α, leading to higher phosphorylated eIF2α (P-eIF2α) levels and reduced translation initiation during the subjective day. This daytime activation of CPC-3 is driven by its binding to uncharged tRNA, and uncharged tRNA levels peak during the day under control of the circadian clock. The daily rhythm in uncharged tRNA levels could arise from rhythmic amino acid levels or aminoacyl-tRNA synthetase (aaRSs) levels. Methods: To determine if and how the clock potentially controls rhythms in aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS), both observed to be rhythmic in circadian genomic datasets, transcriptional and translational fusions to luciferase were generated. These luciferase reporter fusions were examined in wild type (WT), clock mutant Δ frq, and clock-controlled transcription factor deletion strains. Results: Translational and transcriptional fusions of AspRS and GlnRS to luciferase confirmed that their protein levels are clock-controlled with peak levels at night. Moreover, clock-controlled transcription factors NCU00275 and ADV-1 drive robust rhythmic protein expression of AspRS and GlnRS, respectively. Conclusions: These data support a model whereby coordinate clock control of select aaRSs drives rhythms in uncharged tRNAs, leading to rhythmic CPC-3 activation, and rhythms in translation of specific mRNAs.

Circadian clock control of tRNA synthetases in Neurospora crassa

Background: In Neurospora crassa, the circadian clock controls rhythmic mRNA translation initiation through regulation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2). Active CPC-3 phosphorylates and inactivates eIF2α, leading to higher phosphorylated eIF2α (P-eIF2α) levels and reduced translation initiation during the subjective day. This daytime activation of CPC-3 is driven by its binding to uncharged tRNA, and uncharged tRNA levels peak during the day under control of the circadian clock. The daily rhythm in uncharged tRNA levels could arise from rhythmic amino acid levels or aminoacyl-tRNA synthetase (aaRSs) levels. Methods: To determine if and how the clock potentially controls rhythms in aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS), both observed to be rhythmic in circadian genomic datasets, transcriptional and translational fusions to luciferase were generated. These luciferase reporter fusions were examined in wild type (WT), clock mutant Δfrq, and clock-controlled transcription factor deletion strains. Results: Translational and transcriptional fusions of AspRS and GlnRS to luciferase confirmed that their protein levels are clock-controlled with peak levels at night. Moreover, clock-controlled transcription factors NCU00275 and ADV-1 drive robust rhythmic protein expression of AspRS and GlnRS, respectively. Conclusions: These data support a model whereby coordinate clock control of select aaRSs drives rhythms in uncharged tRNAs, leading to rhythmic CPC-3 activation, and rhythms in translation of specific mRNAs.

Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)–like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip–ERS–QRS) and the M-complex (tRip–ERS–MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host–pathogen interaction is discussed.

A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic.

The Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform for genetically encoding new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed to engineer the platform, have impeded its success in the past. Recently, using a facile E. coli-based selection system, we showed that EcTyrRS could be engineered in a strain where the endogenous tyrosyl pair was substituted with an archaeal counterpart. However, significant cross-reactivity between the UAG-suppressing tRNACUAEcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyr that overcomes this cross-reactivity. Optimized selection systems based on this tRNA enabled the efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporated useful ncAAs in mammalian cells, including photoaffinity probes, bioconjugation handles, and a nonhydrolyzable mimic of phosphotyrosine.

Activation of Calpain Contributes to Mechanical Ventilation-Induced Depression of Protein Synthesis in Diaphragm Muscle.

Mechanical ventilation (MV) is a clinical tool that provides respiratory support to patients unable to maintain adequate alveolar ventilation on their own. Although MV is often a life-saving intervention in critically ill patients, an undesired side-effect of prolonged MV is the rapid occurrence of diaphragmatic atrophy due to accelerated proteolysis and depressed protein synthesis. Investigations into the mechanism(s) responsible for MV-induced diaphragmatic atrophy reveal that activation of the calcium-activated protease, calpain, plays a key role in accelerating proteolysis in diaphragm muscle fibers. Moreover, active calpain has been reported to block signaling events that promote protein synthesis (i.e., inhibition of mammalian target of rapamycin (mTOR) activation). While this finding suggests that active calpain can depress muscle protein synthesis, this postulate has not been experimentally verified. Therefore, we tested the hypothesis that active calpain plays a key role in the MV-induced depression of both anabolic signaling events and protein synthesis in the diaphragm muscle. MV-induced activation of calpain in diaphragm muscle fibers was prevented by transgene overexpression of calpastatin, an endogenous inhibitor of calpain. Our findings indicate that overexpression of calpastatin averts MV-induced activation of calpain in diaphragm fibers and rescues the MV-induced depression of protein synthesis in the diaphragm muscle. Surprisingly, deterrence of calpain activation did not impede the MV-induced inhibition of key anabolic signaling events including mTOR activation. However, blockade of calpain activation prevented the calpain-induced cleavage of glutaminyl-tRNA synthetase in diaphragm fibers; this finding is potentially important because aminoacyl-tRNA synthetases play a central role in protein synthesis. Regardless of the mechanism(s) responsible for calpain’s depression of protein synthesis, these results provide the first evidence that active calpain plays an important role in promoting the MV-induced depression of protein synthesis within diaphragm fibers.

Identification and characterization of a fungal-selective glutaminyl tRNA synthetase inhibitor with potent activity against Candida albicans

Candida albicans is the leading cause of systemic candidiasis. Effective treatment is threatened by a dearth of antifungal options and the emergence of resistance. Thus, there is an urgent need to identify novel therapeutic targets to expand our antifungal armamentarium. A promising approach is the discovery of essential genes, as most antimicrobials target essential bioprocesses. Despite detailed characterization of gene essentiality in Saccharomyces cerevisiae,defining essential targets in the pathogen of interest is necessary due to the high level of divergence between these organisms. Thus, using a machine learning algorithm we generated a comprehensive prediction of all genes essential in C. albicans. We leveraged our essentiality predictions with high-throughput screening and chemogenomic datasets to assign the mechanism of action of a previously uncharacterized compound. We identified T-035897 as a molecule with potent bioactivity against C. albicans. Prior chemogenomic profiling in S. cerevisiae suggested that T-035897 targets the glutaminyl tRNA synthetase Gln4, whose homolog in C. albicans was predicted and verified to be required for viability. To confirm the mechanism of T-035897 in C. albicans, we performed haploinsufficiency profiling,which supported Gln4as the target. In parallel, selection of resistant mutants and targeted sequencing uncovered substitutions in the Gln4 catalytic domain. Moreover, T-035897 inhibited translation in afluorescence-based reporter assay. Finally, T-035897 selectively abrogated fungal cell growth in a co-culture model with mammalian cells. Thus, we highlight the power of leveraging essentiality datasets in order to characterize compounds with potent antifungal activity in an effort to unveil novel therapeutic strategies.

Leveraging machine learning essentiality predictions and chemogenomic interactions to identify antifungal targets

Fungal pathogens pose a global threat to human health, with Candida albicans among the leading killers. Systematic analysis of essential genes provides a powerful strategy to discover potential antifungal targets. Here, we build a machine learning model to generate genome-wide gene essentiality predictions for C. albicans and expand the largest functional genomics resource in this pathogen (the GRACE collection) by 866 genes. Using this model and chemogenomic analyses, we define the function of three uncharacterized essential genes with roles in kinetochore function, mitochondrial integrity, and translation, and identify the glutaminyl-tRNA synthetase Gln4 as the target of N-pyrimidinyl-β-thiophenylacrylamide (NP-BTA), an antifungal compound.

Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex.

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.

Glutamyl- and Glutaminyl-tRNA Synthetases Are a Promising Target for the Design of an L-Threonine–Producing Strain

The present work describes an approach that uses a reduction in biomass accumulation during fermentation to improve the properties of a strain producing L-threonine. Glutamyl- and glutaminyl-tRNA synthetases were chosen as targets. Mutants carrying temperature-sensitive alleles of the mentioned enzymes were obtained. It was shown with this system that suppression of the function of tRNA synthetases led to the rapid arrest of culture growth and an increase in the productivity and conversion of L-threonine synthesis. One of the temperature-sensitive strains was used to obtain mutants with the ts phenotype under nonpermissive conditions. Some of these mutants accumulated less biomass and produced 10–12% more threonine than the original strain.

The tRNA‐like small noncoding RNA mascRNA promotes global protein translation

mascRNA is a small cytoplasmic RNA derived from the lncRNA MALAT1. After being processed by the tRNA processing enzymes RNase P and RNase Z, mascRNA undergoes CCA addition like tRNAs and folds into a tRNA-like cloverleaf structure. While MALAT1 functions in multiple cellular processes, the role of mascRNA was largely unknown. Here, we show that mascRNA binds directly to the multi-tRNA synthetase complex (MSC) component glutaminyl-tRNA synthetase (QARS). mascRNA promotes global protein translation and cell proliferation by positively regulating QARS protein levels. Our results uncover a role of mascRNA that is independent of MALAT1, but could be part of the molecular mechanism of MALAT1's function in cancer, and providea paradigm for understanding tRNA-like structures in mammalian cells.

The phylogenetic distribution of the glutaminyl-tRNA synthetase and Glu-tRNAGln amidotransferase in the fundamental lineages would imply that the ancestor of archaea, that of eukaryotes and LUCA were progenotes

The function of the glutaminyl-tRNA synthetase and Glu-tRNAGln amidotransferase might be related to the origin of the genetic code because, for example, glutaminyl-tRNA synthetase catalyses the fundamental reaction that makes the genetic code. If the evolutionary stage of the origin of these two enzymes could be unambiguously identified, then the genetic code should still have been originating at that particular evolutionary stage because the fundamental reaction that makes the code itself was still evidently evolving. This would result in that particular evolutionary moment being attributed to the evolutionary stage of the progenote because it would have a relationship between the genotype and the phenotype not yet fully realized because the genetic code was precisely still originating. I then analyzed the distribution of the glutaminyl-tRNA synthetase and Glu-tRNAGln aminodotrasferase in the main phyletic lineages. Since in some cases the origin of these two enzymes can be related to the evolutionary stages of ancestors of archaea and eukaryotes, this would indicate these ancestors as progenotes because at that evolutionary moment the genetic code was evidently still evolving, thus realizing the definition of progenote. The conclusion that the ancestor of archaea and that of eukaryotes were progenotes would imply that even the last universal common ancestor (LUCA) was a progenote because it appeared, on the tree of life, temporally before these ancestors.

Glutaminyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization, structure, and development as a screening platform.

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl-tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the KM and kcatobs , governing interactions with tRNA, were 1.0 μM and 0.15 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three-dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad-spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild-type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time-kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.

Population analysis of D6-like plasmid prophage variants associated with specific IncC plasmid types in the emerging Salmonella Typhimurium ST213 genotype.

The Salmonella enterica serovar Typhimurium sequence type 213 (ST213) emerged as a predominant genotype in Mexico. It is characterized by harboring multidrug resistance (MDR) IncC plasmids (previously IncA/C) and the lack of the Salmonella virulence plasmid (pSTV). Here we show that the D6-like plasmid prophage is present in most of the ST213 strains. We used the reported nucleotide sequence of YU39 plasmid (pYU39_89) to design a PCR typing scheme for the D6-like plasmid prophages, and determined the complete nucleotide sequences for the D6-like prophages of three additional ST213 strains (YU07-18, SL26 and SO21). Two prophage variants were described: i) a complete prophage, containing homologous sequences for most of the genetic modules described in P1 and D6 phages, which most likely allow for the lytic and lysogenic lifestyles; and ii) an incomplete prophage, lacking a 15 kb region containing morphogenesis genes, suggesting that it is defective. The tail fiber gene inversion region was the most divergent one between D6 and pYU39_89 genomes, suggesting the production of a distinct set of tail fibers, which could be involved in host range preferences. A glutaminyl-tRNA synthetase gene (glnS), which could be involved in providing host cell increased fitness or plasmid maintenance functions, was found in all D6-like genomes. Population level analysis revealed a biogeographic pattern of distribution of these plasmid-phages and specific associations with variants of MDR IncC plasmids. Statistically significant associations were found between the two prophage variants (p75 or p89), the type of IncC plasmids (I or II) and geographic isolation regions (Sonora, San Luis Potosí, Michoacán and Yucatán). This work integrates results from molecular typing, genomics and epidemiology to provide a broad overview for the evolution of an emergent Salmonella genotype.

Conformational changes in glutaminyl-tRNA synthetases upon binding of the substrates and analogs using molecular docking and molecular dynamics approaches

Aminoacyl-tRNA synthetases (aaRSs) are considered as important components in protein translation as they facilitate the attachment of specific transfer RNA (tRNA) to form aminoacyl-tRNAs. Our study focused on understanding the crystal structure of Glutaminyl-tRNA synthetase (GlnRS) from Thermus thermophilus HB8 (PDB ID:5ZDO) and mechanism of formation of enzyme–substrate complex using substrates and its analogs by applying molecular dynamics simulation (MDS) to investigate the conformational changes. Least energy structure of TtGlnRS was considered to dock the enzyme substrates such as glutamine (Gln), glutamic acid (Glu), adenosine monophosphate (AMP), adenosine triphosphate (ATP), QSI and various substrate analogs (2MA, 4SU and 5MU) onto the active site of the enzyme. We focused on comparative analysis of binding specificity between Gln and Glu; similarly, ATP and AMP. Active site organization as observed by MDS analysis showed interactive changes associated with substrate and catalytically important loops. Study found that when tRNAGln specific for GlnRS was docked into the active site of the TtGlnRS enzyme it interacts with 2' OH on the ribose acceptor end of the tRNA. Upon validation with 50 ns MDS, the maximum deviations and conformational changes of secondary structural elements were observed to be high in the loop regions of enzyme–substrate complexes. Binding affinity of ATP to TtGlnRS was further proved by isothermal titration calorimetry.AbbreviationsaaRSsaminoacyl-tRNA synthetasesAMPadenosine monophosphateATPadenosine triphosphateGlideGrid-based LIgand Docking with EnergeticGlnRSglutaminyl-tRNA synthetaseGRAVYGRand AVerage of hydropathicitYGROMACSGROingen Machine for Chemical SimulationsHADDOCKHigh Ambiguity Driven protein–protein DOCKingITCisothermal titration calorimetry2MA2-methyladenosine 5'-(dihydrogen phosphate)MDSmolecular dynamics simulation5MU5-methyluridine 5'-monophosphateNPTnumber of particles, pressure and temperatureNVTnumber of particles, volume and temperatureOPLS-AAoptimized potential for liquid simulation all atomPDBBrookhaven Protein DatabankPMEParticle-Mesh EwaldQSI5'-o-[n-(l-Glutaminyl)-sulfamoyl]adenosineRgradius of gyrationRMSDroot mean square deviationRMSFroot mean square fluctuation4SU4-thiouracil 5'-monophosphateSPCsimple point chargetRNAtransfer ribo nucleic acidTtThermus thermophilusXPextra precisionCommunicated by Ramaswamy H. Sarma

Identification of Inhibitors of Glutaminyl‐tRNA synthetase from Pseudomonas aeruginosa

IntroductionAntibiotic resistance is a growing global threat and infections caused by resistant bacteria have become increasingly difficult to treat with most current antibiotics. Pseudomonas aeruginosa is a Gram‐negative opportunistic pathogen and is the leading cause of mortality in cystic fibrosis patients. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. The glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was over‐expressed, enzymatically characterized and developed into a screening platform for the discovery of chemical compounds that inhibit protein synthesis.MethodsThe P. aeruginosa GlnRS was developed, using scintillation proximity assay (SPA) technology, as a platform to screen chemical compounds for detection of compounds that inhibited function. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with inhibitory activity.ResultsFrom this screening campaign three compounds (BM02E04, BM04B05, and BM04H03) were identified and confirmed as inhibitors of the activity of P. aeruginosa GlnRS. BM02E04, BM04B05, and BM04H03 were observed to inhibit the activity of GlnRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. The minimum inhibitory concentration (MICs) was determined against a panel of bacteria including; E. coli, E. coli tolC mutant, E. faecalis, H. influenzae, M. cattarrhalis, P. aeruginosa, P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain, S. aureus, and S. pneumoniae. There was no activity observed against the wild‐type strain of E. coli or P. aeruginosa, but broad spectrum activity was observed against other Gram‐negative and Gram‐positive bacteria. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus and M. catarrhalis. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, there was no toxicity observed by either BM02E04 or BM04B05 at any concentration tested up to 400 μg/ml.ConclusionP. aeruginosa GlnRS was cloned, over‐expressed, characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms. Three compounds were identified as having inhibitory activity against the aminoacylation activity of GlnRS. Two of the compounds were further characterized for inhibition of enzymatic activity, bacterial growth and toxic effects in human cell cultures.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Глутамил- и глутаминил-тРНК-синтетаза - перспективные мишени для создания штамма-продуцента L-треонина

The present work describes an approach that improves the properties of the strain producing L-threonine via the reduction in the biomass accumulation during fermentation. Glutamyl- and glutaminyl-tRNA synthetases were chosen as targets. Mutants carrying temperature-sensitive alleles were obtained. It was shown that the used system caused the suppression of the function of tRNA synthetases which led to a rapid arrest of the culture growth, and an increase in productivity and yield of the L-threonine synthesis. One of the temperature-sensitive strains was used to obtain under non-permissive conditions of mutants with the suppressed above phenotype. Some of these mutants accumulate less biomass and produce by 10-12% more threonine than the original strain. Escherichia coli, producing strain, threonine, aminoacyl-tRNA synthetase, ts-mutation This work was supported by the Ministry of Science and Higher Education of the Russian Federation (project code RFMEFI61017X0011), and it was carried out using the equipment of the National Bio-Resource Center All-Russian Collection of Industrial Microorganisms, NRC «Kurchatov Institute» - GosNIIgenetika.