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Aminoacyl-tRNA synthetases (aaRSs), essential components of the protein synthesizing machinery, have been often chosen for devising therapeutics against parasitic diseases. Due to their relevance in drug development, the current study was designed to explore functional and structural aspects of Leishmania donovani glutamyl-tRNA synthetase (LdGluRS). Hence, LdGluRS was cloned into an expression vector and purified to homogeneity using chromatographic techniques. Purified protein showed maximum enzymatic activity at physiological pH, with more binding capacity towards its cofactor (Adenosine triphosphate, 0.06 ± 0.01 mM) than the cognate substrate (L-glutamate, 9.5 ± 0.5 mM). Remarkably, salicylate inhibited LdGluRS competitively with respect to L-glutamate and exhibited druglikeness with negligible effect on human macrophages. The protein possessed more α-helices (43 %) than β-sheets (12 %), whereas reductions in thermal stability and cofactor-binding affinity, along with variation in mode of inhibition after mutation signified the role of histidine (H60) as a catalytic residue. LdGluRS could also generate a pro-inflammatory milieu in human macrophages by upregulating cytokines. The docking study demonstrated the placement of salicylate into LdGluRS substrate-binding site, and the complex was found to be stable during molecular dynamics (MD) simulation. Altogether, our study highlights the understanding of molecular inhibition and structural features of glutamyl-tRNA synthetase from kinetoplastid parasites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA. Here we report a molten-globule-like intermediate of HipA at low urea (∼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in-out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be ∼2-2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein translation machinery that provide the charged tRNAs needed for protein synthesis. Over the past decades, aaRSs have been studied as anti-parasitic, anti-bacterial, and anti-fungal drug targets. This study focused on the cytoplasmic glutamyl-tRNA synthetase (GluRS) from Plasmodium falciparum, which belongs to class Ib in aaRSs. GluRS unlike most other aaRSs requires tRNA to activate its cognate amino acid substrate L-Glutamate (L-Glu), and fails to form an intermediate adenylate complex in the absence of tRNA. The crystal structures of the Apo, ATP, and ADP-bound forms of Plasmodium falciparum glutamyl-tRNA synthetase (PfGluRS) were solved at 2.1 Å, 2.2 Å, and 2.8 Å respectively. The structural comparison of the Apo- and ATP-bound holo-forms of PfGluRS showed considerable conformational changes in the loop regions around the ATP-binding pocket of the enzyme. Biophysical characterization of the PfGluRS showed binding of the enzyme substrates L-Gluand ATP.. The sequence and structural conservation were evident across GluRS compared to other species. The structural dissection of the PfGluRS gives insight into the critical residues involved in the binding of ATP substrate, which can be harvested to develop new antimalarial drugs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022. Published by Elsevier B.V.)

The existence of chlorine-resistant bacteria (CRB) in drinking water supply systems (DWSSs) results in significant challenges to the biological security of drinking water. However, little is known about the intrinsic chlorine-resistant molecular metabolic mechanism of bacteria in DWSSs. This research explored the microbial interactions and the key metabolic pathways that modulate the chlorine resistance of bacteria in full-scale chloraminated DWSSs. The dominant CRB, including Bdellovibrio, Bradyrhizobium, Peredibacter, Sphingomonas, and Hydrogenophaga, strongly interacted with each other to maintain basic metabolism. A total of 4.21% of the bacterial metabolic pathways were key and specific to chlorine-resistant bacteria. Glutaminyl-tRNA biosynthesis was the dominant metabolic pathway of CRB in the target DWSSs. After chloramine disinfection, the relative abundance of glutamate-tRNA ligase (GlnRS) and the related orthologous genes increased by 10.11% and 14.58%, respectively. The inactivation rate of the GlnRS overexpression strain (81.40%) was lower than that of the wild-type strain (90.11%) after exposure to chloramine. Meanwhile, the growth rate of the GlnRS overexpression strain was higher than that of the wild-type strain. Glutaminyl-tRNA biosynthesis can enhance chlorine resistance in DWSSs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aminoacylation reaction is the first step of protein biosynthesis. Transfer RNA (tRNA) is charged with an amino acid in this reaction and the reaction is catalyzed by aminoacyl tRNA synthetase enzyme (aaRS). In the present work, we use classical molecular dynamics simulation to show that the tRNA bound Mg 2+ ions significantly influence the charging step of class I Tt GluRS: Glu-AMP: tRNA Glu and class II dimeric Tt SerRS: Ser-AMP: tRNA Ser . The CCA end of the acceptor terminal is disordered in the absence of coordinated Mg 2+ ions and the CCA end can freely explore beyond the specific conformational space of the tRNA in its precharging state. A balance between the conformational disorder of the tRNA and the restriction imposed on the CCA terminal via coordination with the Mg 2+ ions is needed for the placement of the CCA terminal in a precharging state organization. This result provides a molecular-level explanation of the experimental observation that the presence of Mg 2+ ions is a necessary condition for a successful aminoacylation reaction.Communicated by Ramaswamy H. Sarma.

Elizabethkingia bacteria are globally emerging pathogens that cause opportunistic and nosocomial infections, with up to 40% mortality among the immunocompromised. Elizabethkingia species are in the pipeline of organisms for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID). These efforts include the structure-function analysis of potential therapeutic targets. Glutamyl-tRNA synthetase (GluRS) is essential for tRNA aminoacylation and is under investigation as a bacterial drug target. The SSGCID produced, crystallized and determined high-resolution structures of GluRS from E. meningosepticum (EmGluRS) and E. anopheles (EaGluRS). EmGluRS was co-crystallized with glutamate, while EaGluRS is an apo structure. EmGluRS shares ∼97% sequence identity with EaGluRS but less than 39% sequence identity with any other structure in the Protein Data Bank. EmGluRS and EaGluRS have the prototypical bacterial GluRS topology. EmGluRS and EaGluRS have similar binding sites and tertiary structures to other bacterial GluRSs that are promising drug targets. These structural similarities can be exploited for drug discovery., (open access.)

Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNA Glu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics., Competing Interests: Conflict of interest R. C. W. serves as a scientific advisor to HiberCell. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Glutamyl-tRNA synthetase 2 (encoded by EARS2) is a mitochondrial aminoacyl-tRNA synthetase required to translate the 13 subunits of the electron transport chain encoded by the mitochondrial DNA. Pathogenic EARS2 variants cause combined oxidative phosphorylation deficiency, subtype 12 (COXPD12), an autosomal recessive disorder involving lactic acidosis, intellectual disability, and other features of mitochondrial compromise. Patients with EARS2 deficiency present with variable phenotypes ranging from neonatal lethality to a mitigated disease with clinical improvement in early childhood. Here, we report a neonate homozygous for a rare pathogenic variant in EARS2 (c.949G>T; p.G317C). Metabolomics in primary fibroblasts from this patient revealed expected abnormalities in TCA cycle metabolites, as well as numerous changes in purine, pyrimidine, and fatty acid metabolism. To examine genotype-phenotype correlations in COXPD12, we compared the metabolic impact of reconstituting these fibroblasts with wild-type EARS2 versus four additional EARS2 variants from COXPD12 patients with varying clinical severity. Metabolomics identified a group of signature metabolites, mostly from the TCA cycle and amino acid metabolism, that discriminate between EARS2 variants causing relatively mild and severe COXPD12. Taken together, these findings indicate that metabolomics in patient-derived fibroblasts may help establish genotype-phenotype correlations in EARS2 deficiency and likely other mitochondrial disorders., (© 2021 SSIEM.)

The EARS2 nuclear gene encodes mitochondrial glutamyl-tRNA synthetase, a member of the class I family of aminoacyl-tRNA synthetases (aaRSs) that plays a crucial role in mitochondrial protein biosynthesis by catalyzing the charging of glutamate to mitochondrial tRNA(Glu). Pathogenic EARS2 variants have been associated with a rare mitochondrial disorder known as leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The targeted sequencing of 150 nuclear genes encoding respiratory chain complex subunits and proteins implicated in the oxidative phosphorylation (OXPHOS) function was performed. The oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), were measured. The enzymatic activities of Complexes I-V were analyzed spectrophotometrically. We describe a patient carrying two heterozygous EARS2 variants, c.376C>T (p.Gln126*) and c.670G>A (p.Gly224Ser), with infantile-onset disease and a severe clinical presentation. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts, suggesting the molecular mechanism underlying the pathogenicity of these EARS2 variants. Experimental validation using patient-derived fibroblasts allowed an accurate characterization of the disease-causing variants, and by comparing our patient's clinical presentation with that of previously reported cases, new clinical and radiological features of LTBL were identified, expanding the clinical spectrum of this disease.

Antibiotic persistence is a transient phenotypic state during which a bacterium can withstand otherwise lethal antibiotic exposure or environmental stresses. In Escherichia coli, persistence is promoted by the HipBA toxin-antitoxin system. The HipA toxin functions as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neutralizes the toxin. E. coli HipA inactivates the glutamyl-tRNA synthetase GltX, which inhibits translation and triggers the highly conserved stringent response. Although hipBA operons are widespread in bacterial genomes, it is unknown if this mechanism is conserved in other species. Here we describe the functions of three hipBA modules in the alpha-proteobacterium Caulobacter crescentus. The HipA toxins have different effects on growth and macromolecular syntheses, and they phosphorylate distinct substrates. HipA 1 and HipA 2 contribute to antibiotic persistence during stationary phase by phosphorylating the aminoacyl-tRNA synthetases GltX and TrpS. The stringent response regulator SpoT is required for HipA-mediated antibiotic persistence, but persister cells can form in the absence of all hipBA operons or spoT, indicating that multiple pathways lead to persister cell formation in C. crescentus.

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNA Asn via Asp-tRNA Asn . This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNA Asp . Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNA Glu , without producing misacylated tRNAs like Glu-tRNA Asn , Glu-tRNA Asp , or Asp-tRNA Glu , thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation., Competing Interests: The authors declare no conflict of interest

About 1 billion years ago, in a single-celled holozoan ancestor of all animals, a gene fusion of two tRNA synthetases formed the bifunctional enzyme, glutamyl-prolyl-tRNA synthetase (EPRS). We propose here that a confluence of metabolic, biochemical, and environmental factors contributed to the specific fusion of glutamyl- (ERS) and prolyl- (PRS) tRNA synthetases. To test this idea, we developed a mathematical model that centers on the precursor-product relationship of glutamic acid and proline, as well as metabolic constraints on free glutamic acid availability near the time of the fusion event. Our findings indicate that proline content increased in the proteome during the emergence of animals, thereby increasing demand for free proline. Together, these constraints contributed to a marked cellular depletion of glutamic acid and its products, with potentially catastrophic consequences. In response, an ancient organism invented an elegant solution in which genes encoding ERS and PRS fused to form EPRS , forcing coexpression of the two enzymes and preventing lethal dysregulation. The substantial evolutionary advantage of this coregulatory mechanism is evidenced by the persistence of EPRS in nearly all extant animals., (© 2018 Eswarappa et al.)

The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases. We overproduced HipA and HipA7 in Escherichia coli and identified their endogenous substrates by SILAC-based quantitative phosphoproteomics. We confirmed that GltX was the main substrate of both kinase variants and likely the primary determinant of persistence. When HipA and HipA7 were moderately overproduced from plasmids, HipA7 targeted only GltX, but HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation, SeqA. HipA7 showed reduced kinase activity compared to HipA and targeted a substrate pool similar to that of HipA only when produced from a high-copy number plasmid. The kinase variants also differed in autophosphorylation, which was substantially reduced for HipA7. When produced endogenously from the chromosome, HipA showed no activity because of inhibition by the antitoxin HipB, whereas HipA7 phosphorylated GltX and phage shock protein PspA. Initial testing did not reveal a connection between HipA-induced phosphorylation of RplK and persistence or growth inhibition, suggesting that other HipA-specific substrates were likely responsible for growth inhibition. Our results contribute to the understanding of HipA7 action and present a resource for elucidating HipA-related persistence., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Aminoacyl-tRNA synthetases (aaRSs) have housekeeping roles in protein synthesis, but little is known about how these aaRSs are involved in organ development. Here, we report that a rice ( Oryza sativa ) glutamyl-tRNA synthetase (OsERS1) maintains proper somatic cell organization and limits the overproliferation of male germ cells during early anther development. The expression of OsERS1 is specifically detectable in meristematic layer 2-derived cells of the early anther, and osers1 anthers exhibit overproliferation and disorganization of layer 2-derived cells, producing fused lobes and extra germ cells in early anthers. The conserved biochemical function of OsERS1 in ligating glutamate to tRNA Glu is enhanced by its cofactor aaRS OsARC. Furthermore, metabolomics profiling revealed that OsERS1 is an important node for multiple metabolic pathways, indicated by the accumulation of amino acids and tricarboxylic acid cycle components in osers1 anthers. Notably, the anther defects of the osers1 mutant are causally associated with the abnormal accumulation of hydrogen peroxide, which can reconstitute the osers1 phenotype when applied to wild-type anthers. Collectively, these findings demonstrate how aaRSs affect male organ development in plants, likely through protein synthesis, metabolic homeostasis, and redox status., (© 2018 American Society of Plant Biologists. All rights reserved.)

Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is a recently described autosomal recessive mitochondrial disease characterized by early onset of neurological symptoms, a biphasic clinical course, and distinctive neuroimaging. Pathogenic variants in the EARS2 gene that encode for mitochondrial glutamyl-tRNA synthetase are responsible for LTBL. Here, we describe the clinical course of an infant diagnosed with an acute crisis of LTBL and severe liver disease. This article illustrates the utility of blood lactate quantification in addition to basic metabolic testing and brain imaging in a child with low tone and poor growth. In addition, this case demonstrates the utility of current genetic diagnostic testing, in lieu of more invasive procedures, in obtaining rapid answers in this very complicated group of disorders., (Georg Thieme Verlag KG Stuttgart · New York.)

Mitochondrial glutamyl-tRNA synthetase is a major component of protein biosynthesis that loads tRNAs with cognate amino acids. Mutations in the gene encoding this enzyme have been associated with a variety of disorders related to oxidative phosphorylation. Here, we present a case of leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) presenting a biphasic clinical course characterized by delayed psychomotor development and seizure. High-throughput sequencing revealed a novel compound heterozygous mutation in mitochondrial glutamyl-tRNA synthetase 2 (EARS2), which appears to be causative of disease symptoms., (Copyright © 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Arc1p is a yeast-specific tRNA-binding protein that forms a ternary complex with glutamyl-tRNA synthetase (GluRSc) and methionyl-tRNA synthetase (MetRS) in the cytoplasm to regulate their catalytic activities and subcellular distributions. Despite Arc1p not being involved in any known biotin-dependent reaction, it is a natural target of biotin modification. Results presented herein show that biotin modification had no obvious effect on the growth-supporting activity, subcellular distribution, tRNA binding, or interactions of Arc1p with GluRSc and MetRS. Nevertheless, biotinylation of Arc1p was temperature dependent; raising the growth temperature from 30 to 37 °C drastically reduced its biotinylation level. As a result, Arc1p purified from a yeast culture that had been grown overnight at 37 °C was essentially biotin free. Non-biotinylated Arc1p was more heat stable, more flexible in structure, and more effective than its biotinylated counterpart in promoting glutamylation activity of the otherwise inactive GluRSc at 37 °C in vitro Our study suggests that the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Leukoencephalopathy with thalamus and brainstem involvement, and high lactate (LTBL) is a recently identified disease related to mutations in the EARS2 gene encoding glutamyl-tRNA synthetase. We report clinical and radiological findings for two siblings with new pathogenic mutations in the EARS2 gene. Both patients showed symptoms of mild-type disease, but there were clinical differences between the two siblings. While the older brother had hypotonia and delayed developmental milestones, the younger brother had seizures and spasticity in the lower extremities. Brain magnetic resonance imaging (MRI) findings were quite similar for the two siblings. MRI findings were specific to LTBL. MRI lesions of the older sibling had regressed over time. Clinical and radiological improvement, as in the previously reported patients with LTBL, may be an important clue for diagnosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Childhood leukoencephalopathies are a broad class of diseases, which are extremely rare. The treatment and classification of these disorders are both challenging. Nearly half of children presenting with a leukoencephalopathy remain without a specific diagnosis. Leukoencephalopathy with thalamus and brain stem involvement and high lactate (LTBL) is a newly described childhood leukoencephalopathy caused by mutations in the gene encoding a mitochondrial aminoacyl-tRNA synthetase specific for glutamate, EARS2 Magnetic resonance images show a characteristic leukoencephalopathy with thalamic and brain stem involvement. Here, we report a different clinical course of LTBL supported by typical MRI features in a Turkish patient who presented with a history of failure to walk. The EARS2 gene mutation analysis identified a c.322C>T transition, predicting a p.R108W change. This is the first reported early-onset mild type LTBL caused by a homozygous EARS2 mutation case in the literature., (© The Author(s) 2016.)

Mitochondrial aminoacyl tRNA synthetases are essential for organelle protein synthesis. Genetic defects affecting the function of these enzymes may cause pediatric mitochondrial disease. Here, we report on a child with fatal neonatal lactic acidosis and recurrent hypoglycemia caused by mutations in EARS2, encoding mitochondrial glutamyl-tRNA synthetase 2. Brain ultrasound revealed agenesis of corpus callosum. Studies on patient-derived skin fibroblasts showed severely decreased EARS2 protein levels, elevated reactive oxygen species (ROS) production, and altered mitochondrial morphology. Our report further illustrates the clinical spectrum of the severe neonatal-onset form of EARS2 mutations. Moreover, in this case the live-cell parameters appeared to be more sensitive to mitochondrial dysfunction compared to standard diagnostics, which indicates the potential relevance of fibroblast studies in children with mitochondrial diseases.

Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is caused by autosomal recessive EARS2 mutations. Onset is most often in infancy, but in severe cases in the neonatal period. Patients typically have magnetic resonance imaging (MRI) signal abnormalities involving the thalamus, brainstem, and deep cerebral white matter. Most signal abnormalities resolve, but in severe cases at the expense of tissue loss. Here, we report a patient with an encephalopathy of antenatal onset. His early MRI at 8 months of age showed signal abnormalities in the deep cerebral white matter that improved over time. The thalami were absent with the configuration of a developmental anomaly, without evidence of a lesion. We hypothesized that this was a case of LTBL in which the thalamic damage occurred antenatally and was incorporated in the normal brain development. The diagnosis was confirmed by a novel homozygous EARS2 mutation. Our case adds to the phenotypic and genetic spectrum of LTBL., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Corynebacterium glutamicum is generally regarded as a safe microorganism and is used to produce many biochemicals, including L-glutamate. 5-Aminolevulinic acid (ALA) is an L-glutamate derived non-protein amino acid, and is widely applied in fields such as medicine and agriculture., Results: The products of the gltX, hemA, and hemL genes participate in the synthesis of ALA from L-glutamate. Their annotated C. glutamicum homologs were shown to be functional using heterologous complementation and overexpression techniques. Coexpression of hemA and hemL in native host led to the accumulation of ALA, suggesting the potential of C. glutamicum to produce ALA for research and commercial purposes. To improve ALA production, we constructed recombinant C. glutamicum strains expressing hemA and hemL derived from different organisms. Transcriptome analysis indicated that the dissolved oxygen level and Fe(2+) concentration had major effects on ALA synthesis. The downstream pathway of heme biosynthesis was inhibited using small molecules or introducing genetic modifications. Small-scale flask cultures of engineered C. glutamicum produced 1.79 g/L of ALA., Conclusion: Functional characterization of the key enzymes indicated complex regulation of the heme biosynthetic pathway in C. glutamicum. Systematic analysis and molecular genetic engineering of C. glutamicum may facilitate its development as a system for large-scale synthesis of ALA.

The putative zinc-binding domain (pZBD) in Escherichia coli glutamyl-tRNA synthetase (GluRS) is known to correctly position the tRNA acceptor arm and modulate the amino acid-binding site. However, its functional role in other bacterial species is not clear since many bacterial GluRSs lack a zinc-binding motif in the pZBD. From experimental studies on pZBD-swapped E. coli GluRS, with Thermosynechoccus elongatus GluRS, Burkholderia thailandensis GluRS and E. coli glutamyl-queuosine-tRNA(Asp) synthetase (Glu-Q-RS), we show that E. coli GluRS, containing the zinc-free pZBD of B. thailandensis, is as functional as the zinc-bound wild-type E. coli GluRS, whereas the other constructs, all zinc-bound, show impaired function. A pZBD-tinkered version of E. coli GluRS that still retained Zn-binding capacity, also showed reduced activity. This suggests that zinc is not essential for the pZBD to be functional. From extensive structural and sequence analyses from whole genome database of bacterial GluRS, we further show that in addition to many bacterial GluRS lacking a zinc-binding motif, the pZBD is actually deleted in some bacteria, all containing either glutaminyl-tRNA synthetase (GlnRS) or a second copy of GluRS (GluRS2). Correlation between the absence of pZBD and the occurrence of glutamine amidotransferase CAB (GatCAB) in the genome suggests that the primordial role of the pZBD was to facilitate transamidation of misacylated Glu-tRNA(Gln) via interaction with GatCAB, whereas its role in tRNA(Glu) interaction may be a consequence of the presence of pZBD.

The nature of interaction between glutamyl-tRNA synthetase (GluRS) and its tRNA substrate is unique in bacteria in that many bacterial GluRS are capable of recognizing two tRNA substrates: tRNAGlu and tRNAGln. To properly understand this distinctive GluRS-tRNA interaction it is important to pursue detailed structure-function studies; however, because of the fact that tRNA-GluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, the structure-function correlation studies must also be phylum-specific. GluRS from Thermus thermophilus and Escherichia coli, which belong to evolutionarily distant phyla, are the biochemically best characterized. Of these, only the structure of T. thermophilus GluRS is available. To fully unravel the subtleties of tRNAGlu-GluRS interaction in E. coli, a model bacterium that can also be pathogenic, determination of the E. coli GluRS structure is essential. However, previous attempts have failed to crystallize E. coli GluRS. By mapping crystal contacts of a homologous GluRS onto the E. coli GluRS sequence, two surface residues were identified that might have been hindering crystallization attempts. Accordingly, these two residues were mutated and crystallization of the double mutant was attempted. Here, the design, expression, purification and crystallization of an engineered E. coli GluRS in which two surface residues were mutated to optimize crystal contacts are reported.

Background: Evolutionary histories of glutamyl-tRNA synthetase (GluRS) and glutaminyl-tRNA synthetase (GlnRS) in bacteria are convoluted. After the divergence of eubacteria and eukarya, bacterial GluRS glutamylated both tRNAGln and tRNAGlu until GlnRS appeared by horizontal gene transfer (HGT) from eukaryotes or a duplicate copy of GluRS (GluRS2) that only glutamylates tRNAGln appeared. The current understanding is based on limited sequence data and not always compatible with available experimental results. In particular, the origin of GluRS2 is poorly understood., Results: A large database of bacterial GluRS, GlnRS, tRNAGln and the trimeric aminoacyl-tRNA-dependent amidotransferase (gatCAB), constructed from whole genomes by functionally annotating and classifying these enzymes according to their mutual presence and absence in the genome, was analyzed. Phylogenetic analyses showed that the catalytic and the anticodon-binding domains of functional GluRS2 (as in Helicobacter pylori) were independently acquired from evolutionarily distant hosts by HGT. Non-functional GluRS2 (as in Thermotoga maritima), on the other hand, was found to contain an anticodon-binding domain appended to a gene-duplicated catalytic domain. Several genomes were found to possess both GluRS2 and GlnRS, even though they share the common function of aminoacylating tRNAGln. GlnRS was widely distributed among bacterial phyla and although phylogenetic analyses confirmed the origin of most bacterial GlnRS to be through a single HGT from eukarya, many GlnRS sequences also appeared with evolutionarily distant phyla in phylogenetic tree. A GlnRS pseudogene could be identified in Sorangium cellulosum., Conclusions: Our analysis broadens the current understanding of bacterial GlxRS evolution and highlights the idiosyncratic evolution of GluRS2. Specifically we show that: i) GluRS2 is a chimera of mismatching catalytic and anticodon-binding domains, ii) the appearance of GlnRS and GluRS2 in a single bacterial genome indicating that the evolutionary histories of the two enzymes are distinct, iii) GlnRS is more widespread in bacteria than is believed, iv) bacterial GlnRS appeared both by HGT from eukarya and intra-bacterial HGT, v) presence of GlnRS pseudogene shows that many bacteria could not retain the newly acquired eukaryal GlnRS. The functional annotation of GluRS, without recourse to experiments, performed in this work, demonstrates the inherent and unique advantages of using whole genome over isolated sequence databases.

The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA(Gln) biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA(Glu) and tRNA(Gln), determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA(Gln) biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.

HipA of Escherichia coli is a eukaryote-like serine-threonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA. Instead, a genetic screen reveals that the overexpression of glutamyl-tRNA synthetase (GltX) suppresses the toxicity of HipA. We show that HipA phosphorylates conserved Ser(239) near the active center of GltX and inhibits aminoacylation, a unique example of an aminoacyl-tRNA synthetase being inhibited by a toxin encoded by a toxin-antitoxin locus. HipA only phosphorylates tRNA(Glu)-bound GltX, which is consistent with the earlier finding that the regulatory motif containing Ser(239) changes configuration upon tRNA binding. These results indicate that HipA mediates persistence by the generation of "hungry" codons at the ribosomal A site that trigger the synthesis of (p)ppGpp, a hypothesis that we verify experimentally., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aminoacyl-tRNA synthetases recognize cognate amino acids and tRNAs from their noncognate counterparts and catalyze the formation of aminoacyl-tRNAs. Halofuginone (HF), a coccidiostat used in veterinary medicine, exerts its effects by acting as a high-affinity inhibitor of the enzyme glutamyl-prolyl-tRNA synthetase (EPRS). In order to elucidate the precise molecular basis of this inhibition mechanism of human EPRS, the crystal structures of the prolyl-tRNA synthetase domain of human EPRS (hPRS) at 2.4 Å resolution (hPRS-apo), of hPRS complexed with ATP and the substrate proline at 2.3 Å resolution (hPRS-sub) and of hPRS complexed with HF at 2.62 Å resolution (hPRS-HF) are presented. These structures show plainly that motif 1 functions as a cap in hPRS, which is loosely opened in hPRS-apo, tightly closed in hPRS-sub and incorrectly closed in hPRS-HF. In addition, the structural analyses are consistent with more effective binding of hPRS to HF with ATP. Mutagenesis and biochemical analysis confirmed the key roles of two residues, Phe1097 and Arg1152, in the HF inhibition mechanism. These structures will lead to the development of more potent and selective hPRS inhibitors for promoting inflammatory resolution.

Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNA(Gln) is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNA(Gln), and an amidotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln). The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNA(Gln). It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.

The genus Brachycybe Wood is a little known group of millipedes comprising eight nominal species distributed throughout North America, Japan, South Korea, Taiwan, and China. The group's species are relatively morphologically homogenous and have been described primarily on the basis of differences in somatic morphology largely ignoring the often-used characters in millipede taxonomy and systematics - male genitalia (the gonopods). The objectives of this study were to survey male gonopods with the aim of evaluating inter-specific variation, assess existing species boundaries and phylogeny using molecular characters, examine the historical biogeography of the genus, and estimate the timing of lineage divergence using a molecular clock. We surveyed two mitochondrial genes (cytochrome c oxidase I and cytochrome b) and one nuclear protein-coding gene (glutamyl and prolyl-tRNA synthetase) each comprising 952, 746, and 555 aligned base pairs respectively. Phylogenetic inference coupled with an analysis of species delimitation using a generalized mixed Yule coalescent model recovered eight species, two of which were considered to be cryptic. Molecular dating analyses coupled with ancestral range reconstructions indicate that the group is quite ancient (age>50 million years) and its origins are likely traced back geographically to the mountains of California's Sierra Nevada and Coastal Ranges. The genus Brachycybe appears to have expanded its range at least twice out of present day California into eastern North America and at least once into Asia. This study highlights the need for integrative approaches to describe biodiversity and furthers the evidence for cryptic diversity even in groups where genitalia are generally thought to be rapidly evolving, diagnostic features., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nucleotides whose mutations seriously affect glutamylation efficiency are experimentally known for Escherichia coli tRNA(Glu). However, not much is known about functional hotspots on the complementary enzyme, glutamyl-tRNA synthetase (GluRS). From structural and functional studies on an Arg266Leu mutant of E. coli GluRS, we demonstrate that Arg266 is essential for efficient glutamylation of tRNA(Glu). Consistent with this result, we found that Arg266 is a conserved signature of proteobacterial GluRS. In contrast, most non-proteobacterial GluRS contain Leu, and never Arg, at this position. Our results imply a unique strategy of glutamylation of tRNA(Glu) in proteobacteria under phylum-specific evolutionary compulsions., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Molten globule and other disordered states of proteins are now known to play important roles in many cellular processes. From equilibrium unfolding studies of two paralogous proteins and their variants, glutaminyl-tRNA synthetase (GlnRS) and two of its variants [glutamyl-tRNA synthetase (GluRS) and its isolated domains, and a GluRS-GlnRS chimera], we demonstrate that only GlnRS forms a molten globule-like intermediate at low urea concentrations. We demonstrated that a loop in the GlnRS C-terminal anticodon binding domain that promotes communication with the N-terminal domain and indirectly modulates amino acid binding is also responsible for stabilization of the molten globule state. This loop was inserted into GluRS in the eukaryotic branch after the archaea-eukarya split, right around the time when GlnRS evolved. Because of the structural and functional importance of the loop, it is proposed that the insertion of the loop into a putative ancestral GluRS in eukaryotes produced a catalytically active molten globule state. Because of their enhanced dynamic nature, catalytically active molten globules are likely to possess broad substrate specificity. It is further proposed that the putative broader substrate specificity allowed the catalytically active molten globule to accept glutamine in addition to glutamic acid, leading to the evolution of GlnRS.

In the large group of genetically undetermined infantile-onset mitochondrial encephalopathies, multiple defects of mitochondrial DNA-related respiratory-chain complexes constitute a frequent biochemical signature. In order to identify responsible genes, we used exome-next-generation sequencing in a selected cohort of patients with this biochemical signature. In an isolated patient, we found two mutant alleles for EARS2, the gene encoding mitochondrial glutamyl-tRNA synthetase. The brain magnetic resonance imaging of this patient was hallmarked by extensive symmetrical cerebral white matter abnormalities sparing the periventricular rim and symmetrical signal abnormalities of the thalami, midbrain, pons, medulla oblongata and cerebellar white matter. Proton magnetic resonance spectroscopy showed increased lactate. We matched this magnetic resonance imaging pattern with that of a cohort of 11 previously selected unrelated cases. We found mutations in the EARS2 gene in all. Subsequent detailed clinical and magnetic resonance imaging based phenotyping revealed two distinct groups: mild and severe. All 12 patients shared an infantile onset and rapidly progressive disease with severe magnetic resonance imaging abnormalities and increased lactate in body fluids and proton magnetic resonance spectroscopy. Patients in the 'mild' group partially recovered and regained milestones in the following years with striking magnetic resonance imaging improvement and declining lactate levels, whereas those of the 'severe' group were characterized by clinical stagnation, brain atrophy on magnetic resonance imaging and persistent lactate increases. This new neurological disease, early-onset leukoencephalopathy with thalamus and brainstem involvement and high lactate, is hallmarked by unique magnetic resonance imaging features, defined by a peculiar biphasic clinical course and caused by mutations in a single gene, EARS2, expanding the list of medically relevant defects of mitochondrial DNA translation.

Objective: Glutamyl tRNA Synthetase (Gts) is a protease which catalyzes esterification between tRNA and glutamine. The immunogenicity of Gts was evaluated through immunization and challenge experiment., Methods and Results: We cloned gts from the genome of SC19, and inserted it into prokaryotic expression plasmid pET28a-gts. The recombinant vector was transformed into E. coli BL21. Induced by IPTG, one 58 kD protein, was expressed and purified by using Ni - NTA column (Novagen). The purity of rGtS was 93.3% and the concentration of purified protein was 433 microg/mL. We proved the immunogenicity of recombinant protein rGtS by western blot analysis. We immunized Balb/c mice with rGtS and Freund's adjuvant, and after two boost vaccinations, all mice were challenged with 4 times LD50 amount of SC19 (1.2 x 10(9) CFU). The survive rate of vaccination group is 50% (4/8), significantly higher than blank vector control group (1/8)., Conclusion: These results proved that GtS has certain immunogenicity and can offer partial protection against high dose challenge. Therefore Gts could be a potential candidate of subunit vaccine against Streptococcus suis.

Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA(Gln) is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA(Gln) to form Glu-tRNA(Gln). In addition, we have identified a human homolog of the Glu-tRNA(Gln) amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA(Gln) accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA(Gln) formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA(Gln) has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA(Gln) is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.

Faithful translation of the genetic code is mainly based on the specificity of tRNA aminoacylation catalyzed by aminoacyl-tRNA synthetases. These enzymes are comprised of a catalytic core and several appended domains. Bacterial glutamyl-tRNA synthetases (GluRS) contain five structural domains, the two distal ones interacting with the anticodon arm of tRNA(Glu). Thermus thermophilus GluRS requires the presence of tRNA(Glu) to bind ATP in the proper site for glutamate activation. In order to test the role of these two distal domains in this mechanism, we characterized the in vitro properties of the C-truncated Escherichia coli GluRSs N(1-313) and N(1-362), containing domains 1-3 and 1-4, respectively, and of their N-truncated complements GluRSs C(314-471) (containing domains 4 and 5) and C(363-471) (free domain 5). These C-truncated GluRSs are soluble, aminoacylate specifically tRNA(Glu), and require the presence of tRNA(Glu) to catalyze the activation of glutamate, as does full-length GluRS(1-471). The k(cat) of tRNA glutamylation catalyzed by N(1-362) is about 2000-fold lower than that catalyzed by the full-length E. coli GluRS(1-471). The addition of free domain 5 (C(363-471)) to N(1-362) strongly stimulates this k(cat) value, indicating that covalent connectivity between N(1-362) and domain 5 is not required for GluRS activity; the hyperbolic relationship between domain 5 concentration and this stimulation indicates that these proteins and tRNA(Glu) form a productive complex with a K(d) of about 100 microM. The K(d) values of tRNA(Glu) interactions with the full-length GluRS and with the truncated GluRSs N(1-362) and free domain 5 are 0.48, 0.11, and about 1.2 microM, respectively; no interaction was detected between these two complementary truncated GluRSs. These results suggest that in the presence of these truncated GluRSs, tRNA(Glu) is positioned for efficient aminoacylation by the two following steps: first, it interacts with GluRS N(1-362) via its acceptor-TPsiC stem loop domain and then with free domain 5 via its anticodon-Dstem-biloop domain, which appeared later during evolution. On the other hand, tRNA glutamylation catalyzed by N(1-313) is not stimulated by its complement C(314-471), revealing the importance of the covalent connectivity between domains 3 and 4 for GluRS aminoacylation activity. The K(m) values of N(1-313) and N(1-362) for each of their substrates are similar to those of full-length GluRS. These C-truncated GluRSs recognize only tRNA(Glu). These results confirm the modular nature of GluRS and support the model of a "recent" fusion of domains 4 and 5 to a proto-GluRS containing the catalytic domain and able to recognize its tRNA substrate(s).

Discrimination of tRNA(Gln) is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA(Gln) and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA(Gln). Our results demonstrate that in addition to the anticodon-binding domain, tRNA(Gln) discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.

The moc3 gene was screened out as an inducer of sexual differentiation in fission yeast Schizosaccharomyces pombe. We isolated a novel gene, named ers2, encoding mitochondrial glutamyl tRNA synthetase (mGluRS) as a Moc3 interacting element by the yeast two-hybrid system. Cytoplasmic glutamyl tRNA synthetase (cGluRS) also interacted with Moc3 in a yeast two-hybrid system. Disruption of ers1 (cGluRS) and of ers2 (mGluRS) indicated that these genes are both essential for the cell growth of S. pombe. We found that ers2 severely affected cell growth and decreased viability, but induced sexual differentiation of S. pombe when it was over-expressed. Over-expression of ers1 also stimulated sexual differentiation in S. pombe. These observations led us to test the effects of various amino acids on sexual differentiation. We found that glutamic acid, as well as other specific amino acids, such as tryptophan, methionine, and threonine, efficiently induced sexual differentiation in S. pombe. Our findings suggest a new regulatory mechanism where GluRSs and glutamic acid are involved in sexual differentiation in S. pombe.

A comparative genomic analysis of 35 cyanobacterial strains has revealed that the gene complement of aminoacyl-tRNA synthetases (AARSs) and routes for aminoacyl-tRNA synthesis may differ among the species of this phylum. Several genes encoding AARS paralogues were identified in some genomes. In-depth phylogenetic analysis was done for each of these proteins to gain insight into their evolutionary history. GluRS, HisRS, ArgRS, ThrRS, CysRS, and Glu-Q-RS showed evidence of a complex evolutionary course as indicated by a number of inconsistencies with our reference tree for cyanobacterial phylogeny. In addition to sequence data, support for evolutionary hypotheses involving horizontal gene transfer or gene duplication events was obtained from other observations including biased sequence conservation, the presence of indels (insertions or deletions), or vestigial traces of ancestral redundant genes. We present evidences for a novel protein domain with two putative transmembrane helices recruited independently by distinct AARS in particular cyanobacteria.

Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K(M) of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA(Gln) by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA(Gln) by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA(Gln) complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.

Glutaminyl-tRNA synthetase and asparaginyl-tRNA synthetase evolved from glutamyl-tRNA synthetase and aspartyl-tRNA synthetase, respectively, after the split in the last universal communal ancestor (LUCA). Glutaminyl-tRNA(Gln) and asparaginyl-tRNA(Asn) were likely formed in LUCA by amidation of the mischarged species, glutamyl-tRNA(Gln) and aspartyl-tRNA(Asn), by tRNA-dependent amidotransferases, as is still the case in most bacteria and all known archaea. The amidotransferase GatCAB is found in both domains of life, while the heterodimeric amidotransferase GatDE is found only in Archaea. The GatB and GatE subunits belong to a unique protein family that includes Pet112 that is encoded in the nuclear genomes of numerous eukaryotes. GatE was thought to have evolved from GatB after the emergence of the modern lines of decent. Our phylogenetic analysis though places the split between GatE and GatB, prior to the phylogenetic divide between Bacteria and Archaea, and Pet112 to be of mitochondrial origin. In addition, GatD appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, while GatDE is an archaeal signature protein, it likely was present in LUCA together with GatCAB. Archaea retained both amidotransferases, while Bacteria emerged with only GatCAB. The presence of GatDE has favored a unique archaeal tRNA(Gln) that may be preventing the acquisition of glutaminyl-tRNA synthetase in Archaea. Archaeal GatCAB, on the other hand, has not favored a distinct tRNA(Asn), suggesting that tRNA(Asn) recognition is not a major barrier to the retention of asparaginyl-tRNA synthetase in many Archaea.

Eukaryotic aminoacyl-tRNA synthetases are usually organized into high-molecular-weight complexes, the structure and function of which are poorly understood. We have previously described a yeast complex containing two aminoacyl-tRNA synthetases, methionyl-tRNA synthetase and glutamyl-tRNA synthetase, and one noncatalytic protein, Arc1p, which can stimulate the catalytic efficiency of the two synthetases. To understand the complex assembly mechanism and its relevance to the function of its components, we have generated specific mutations in residues predicted by a recent structural model to be located at the interaction interfaces of the N-terminal domains of all three proteins. Recombinant wild-type or mutant forms of the proteins, as well as the isolated N-terminal domains of the two synthetases, were overexpressed in bacteria, purified and used for complex formation in vitro and for determination of binding affinities using surface plasmon resonance. Moreover, mutant proteins were expressed as PtA or green fluorescent protein fusion polypeptides in yeast strains lacking the endogenous proteins in order to monitor in vivo complex assembly and their subcellular localization. Our results show that the assembly of the Arc1p-synthetase complex is mediated exclusively by the N-terminal domains of the synthetases and that the two enzymes bind to largely independent sites on Arc1p. Analysis of single-amino-acid substitutions identified residues that are directly involved in the formation of the complex in yeast cells and suggested that complex assembly is mediated predominantly by van der Waals and hydrophobic interactions, rather than by electrostatic forces. Furthermore, mutations that abolish the interaction of methionyl-tRNA synthetase with Arc1p cause entry of the enzyme into the nucleus, proving that complex association regulates its subcellular distribution. The relevance of these findings to the evolution and function of the multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases is discussed.

Aminoacyl-tRNA synthetases are normally found in one of two mutually exclusive structural classes, the only known exception being lysyl-tRNA synthetase which exists in both classes I (LysRS1) and II (LysRS2). Differences in tRNA acceptor stem recognition between LysRS1 and LysRS2 do not drastically impact cellular aminoacylation levels, focusing attention on the mechanism of tRNA anticodon recognition by LysRS1. On the basis of structure-based sequence alignments, seven tRNALys anticodon variants and seven LysRS1 anticodon binding site variants were selected for analysis of the Pyrococcus horikoshii LysRS1-tRNALys docking model. LysRS1 specifically recognized the bases at positions 35 and 36, but not that at position 34. Aromatic residues form stacking interactions with U34 and U35, and aminoacylation kinetics also identified direct interactions between Arg502 and both U35 and U36. Tyr491 was also found to interact with U36, and the Y491E variant exhibited significant improvement compared to the wild type in aminoacylation of a tRNALysUUG mutant. Refinement of the LysRS1-tRNALys docking model based upon these data suggested that anticodon recognition by LysRS1 relies on considerably fewer interactions than that by LysRS2, providing a structural basis for the more significant role of the anticodon in tRNA recognition by the class II enzyme. To date, only glutamyl-tRNA synthetase (GluRS) has been found to contain an alpha-helix cage anticodon binding domain homologous to that of LysRS1, and these data now suggest that specificity for the anticodon of tRNALys could have been acquired through relatively few changes to the corresponding domain of an ancestral GluRS enzyme.

The genome of Tolypothrix sp. PCC 7601 carries two copies of a novel insertion sequence, ISTosp1. One of the two copies is located upstream of the gene encoding glutamyl-tRNA synthetase, an enzyme playing a key role in protein and pigment synthesis. The tnpA gene of the IS element and gltX were co-transcribed and their expression was transiently upregulated upon retrieval of the ammonium source irrespective of whether nitrate or no nitrogen source were available. The second copy is also transcribed and shows a similar regulatory pattern. Structural elements of the promoter (-10 and -35 sequences) directing the expression of the tnpA-gltX operon have been localized within the IS. Regulatory sequences involving the NtcA transcription factor in the control of tnpA-gltX expression were found both within and in sequences upstream of the insertion element. The expression of gltX in a closely related cyanobacterium, Nostoc sp. PCC 7120, which lacks the insertion upstream of gltX, decreased upon ammonium retrieval, a regulatory pattern that markedly differs from that observed in Tolypothrix sp. PCC 7601. ISTosp1 constitutes a good example of how cells can make use of a transposable element to evolve an original regulatory mechanism.

Motivation: Determining orthology relations among genes across multiple genomes is an important problem in the post-genomic era. Identifying orthologous genes can not only help predict functional annotations for newly sequenced or poorly characterized genomes, but can also help predict new protein-protein interactions. Unfortunately, determining orthology relation through computational methods is not straightforward due to the presence of paralogs. Traditional approaches have relied on pairwise sequence comparisons to construct graphs, which were then partitioned into putative clusters of orthologous groups. These methods do not attempt to preserve the non-transitivity and hierarchic nature of the orthology relation., Results: We propose a new method, COCO-CL, for hierarchical clustering of homology relations and identification of orthologous groups of genes. Unlike previous approaches, which are based on pairwise sequence comparisons, our method explores the correlation of evolutionary histories of individual genes in a more global context. COCO-CL can be used as a semi-independent method to delineate the orthology/paralogy relation for a refined set of homologous proteins obtained using a less-conservative clustering approach, or as a refiner that removes putative out-paralogs from clusters computed using a more inclusive approach. We analyze our clustering results manually, with support from literature and functional annotations. Since our orthology determination procedure does not employ a species tree to infer duplication events, it can be used in situations when the species tree is unknown or uncertain., Contact: jothi@mail.nih.gov, przytyck@mail.nih.gov, Supplementary Information: Supplementary materials are available at Bioinformatics online.

We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins containing unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.

Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants.