In Helicobacter pylon, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated GluARNA(Gln) and AspARNA(Asn) into Gln-tRNA(Gln) and Asn-tRNA(Asn), respectively. In this study, we used a phage library to identify peptide inhibitors of GatCAB. A library displaying loop-constrained heptapeptides was used to screen for phages binding to the purified GatCAB. To optimize the probability of obtaining competitive inhibitors of GatCAB with respect to its substrate GluARNA(Gln), we used that purified substrate in the biopanning process of the phage-display technique to elute phages bound to GatCAB at the third round of the biopanning process. Among the eluted phages, we identified several that encode cyclic peptides rich in Trp and Pro that inhibit H. pylon GatCAB in vitro. Peptides P10 and P9 were shown to be competitive inhibitors of GatCAB with respect to its substrate GluARNA(Gln), with K, values of 126 and 392 mu M, respectively. The docking models revealed that the Trp residues of these peptides form pi-pi stacking interactions with Tyr81 of the synthetase active site, as does the 3'-terminal A76 of tRNA, supporting their competitive behavior with respect to GluARNA(Gln) in the transamidation reaction. These peptides can be used as scaffolds in the search for novel antibiotics against the pathogenic bacteria that require GatCAB for Gln-tRNA(Gln) and/or Asn-tRNA(Asn) formation. 2016 Elsevier Inc. All rights reserved.

Glutaminyl-tRNAGln in Helicobacter pylori is formed by an indirect route requiring a noncanonical glutamyl-tRNA synthetase and a tRNA-dependent heterotrimeric amidotransferase (AdT) GatCAB. Widespread use of this pathway among prominent human pathogens, and its absence in the mammalian cytoplasm, identify AdT as a target for the development of antimicrobial agents. We present here the inhibitory properties of three dipeptide-like sulfone-containing compounds analogous to the transamidation intermediates, which are competitive inhibitors of AdT with respect to Glu-tRNAGln . Molecular docking revealed that AdT inhibition by these compounds depends on π-π stacking interactions between their aromatic groups and Tyr81 of the GatB subunit. The properties of these inhibitors indicate that the 3'-terminal adenine of Glu-tRNAGln plays a major role in binding to the AdT transamidation active site.

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

Many enzymes which catalyze the conversion of large substrates are made of several structural domains belonging to the same polypeptide chain. Transfer RNA (tRNA), one of the substrates of the multidomain aminoacyl-tRNA synthetases (aaRS), is an L-shaped molecule whose size in one dimension is similar to that of its cognate aaRS. Crystallographic structures of aaRS/tRNA complexes show that these enzymes use several of their structural domains to interact with their cognate tRNA. This mini review discusses first some aspects of the evolution and of the flexibility of the pentadomain bacterial glutamyl-tRNA synthetase (GluRS) revealed by kinetic and interaction studies of complementary truncated forms, and then illustrates how stable analogues of aminoacyl-AMP intermediates have been used to probe conformational changes in the active sites of Escherichia coli GluRS and of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) of Pseudomonas aeruginosa.

It has been a dream that theoretical biology can be extensively applied in experimental biology to accelerate the understanding of the sophiscated movements in living organisms. A brave assay and an excellent example were represented by enzymology, in which the well-established physico-chemistry is used to describe, to fit, to predict and to improve enzyme reactions. Before the modern bioinformatics, the developments of the combination of theoretical biology and experimental biology have been mainly limited to various classic formulations. The systematic use of graphic rules by Prof. Kuo-Chen Chou and his coworkers has significantly facilitated to deal with complicated enzyme systems. With the recent fast progress of bioinformatics, prediction of protein structures and various protein attributes have been well established by Chou and co-workers, stimulating the experimental biology. For example, their recent method for predicting protein subcellular localization (one of the important attributes of proteins) has been extensively applied by scientific colleagues, yielding many new results with thousands of citations. The research by Prof. Chou is characterized by introducing novel physical concepts as well as powerful and elegant mathematical methods into important biomedical problems, a focus throughout his career, even when facing enormous difficulties. His efforts in 50 years have greatly helped us to realize the dream to make “theoretical and experimental biology in one”. Prof. Richard Giege is well known for his multi-disciplinary research combining physics, chemistry, enzymology and molecular biology. His major focus of study is on the identity of tRNAs and their interactions with aminoacyl-tRNA synthetases (aaRS), which are of critical importance to the fidelity of protein biosynthesis. He and his colleagues have carried out the first crystallization of a tRNA/aaRS complex, that between tRNA and AspRS from yeast. The determination of the complex structure contributed significantly to understand the interaction of protein and RNA. From his fine research, they have also found other biological function of these small RNAs. He has developed in parallel appropriate methods for his research, of which the protein crystallogenesis, a name he has coined, is an excellent example. Now macromolecular crystallogenesis has become a developed science. In fact, such contribution has accelerated the development of protein crystallography, stimulating the study of macromolecular structure and function.

It has been a dream that theoretical biology can be extensively applied in experimental biology to accelerate the understanding of the sophiscated movements in living organisms. A brave assay and an excellent example were represented by enzymology, in which the well-established physico-chemistry is used to describe, to fit, to predict and to improve enzyme reactions. Before the modern bioinformatics, the developments of the combination of theoretical biology and experimental biology have been mainly limited to various classic formulations. The systematic use of graphic rules by Prof. Kuo-Chen Chou and his co-workers has significantly facilitated to deal with complicated enzyme systems. With the recent fast progress of bioinformatics, prediction of protein structures and various protein attributes have been well established by Chou and co-workers, stimulating the experimental biology. For example, their recent method for predicting protein subcellular localization (one of the important attributes of proteins) has been extensively applied by scientific colleagues, yielding many new results with thousands of citations. The research by Prof. Chou is characterized by introducing novel physical concepts as well as powerful and elegant mathematical methods into important biomedical problems, a focus throughout his career, even when facing enormous difficulties. His efforts in 50 years have greatly helped us to realize the dream to make "theoretical and experimental biology in one". Prof. Richard Giege is well known for his multi-disciplinary research combining physics, chemistry, enzymology and molecular biology. His major focus of study is on the identity of tRNAs and their interactions with aminoacyl-tRNA synthetases (aaRS), which are of critical importance to the fidelity of protein biosynthesis. He and his colleagues have carried out the first crystallization of a tRNA/aaRS complex, that between tRNAAsp and AspRS from yeast. The determination of the complex structure contributed significantly to under- stand the interaction of protein and RNA. From his fine research, they have also found other biological function of these small RNAs. He has developed in parallel appropriate methods for his research, of which the protein crystallogenesis, a name he has coined, is an excellent example. Now macromolecular crystallogenesis has become a developed science. In fact, such contribution has accelerated the development of protein crystallography, stimulating the study of macromolecular structure and function.

Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

Only about half of bacterial species use an asparaginyl-tRNA synthetase (AsnRS) to attach Asn to its cognate tRNA(Asn). Other bacteria, including the human pathogen Moraxella catarrhalis, a causative agent of otitis media, lack a gene encoding AsnRS, and form Asn-tRNA(Asn) by an indirect pathway catalysed by two enzymes: first, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) catalyses the formation of aspartyl-tRNA(Asn) (Asp-tRNA(Asn)); then, a tRNA-dependent amidotransferase (GatCAB) transamidates this 'incorrect' product into Asn-tRNA(Asn). As M. catarrhalis has a Gln-tRNA synthetase, its GatCAB functions as an Asp-tRNA(Asn) amidotransferase. This pathogen rapidly evolved to about 90% ampicillin resistance worldwide by insertion of a bro-1 beta-lactamase gene within the gatCAB operon. Comparison of the GatCAB subunits from bro-1 beta-lactamase-positive and bro-negative strains showed that the laterally transferred bro-1 gene, inserted into the gatCAB operon, affected the C-terminal sequence of GatA. The identity between the C-terminal sequences of GatA(wt) (residues 479-491) and of GatA(BRO-1) (residues 479-492) was about 36%, whereas the rest of the GatA sequence was relatively conserved. The characterization of these two distinct GatCABs as well as the hybrid GatCAB containing GatA(1-478)(wt)(479-492)(BRO-1) and truncated GatCAB enzymes of M. catarrhalis showed that the substitution in GatA(wt) of residues 479-492 of GatA(BRO-1) causes increased specificity for glutamine, and decreased specificity for Asp-tRNA(Asn) in the transamidation reaction. We conclude that the bro gene insertion has altered the kinetic parameters of Asp-tRNA(Asn) amidotransferase, and we propose a model for gatA evolution after the insertion of bro-1 at the carboxyl end of gatA.

In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNAAsn and tRNAGln, respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNAGln in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms. This ternary complex containing the non-canonical GluRS2 specific for Glu-tRNAGln formation, the tRNA-dependent amidotransferase GatCAB and tRNAGln was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (KD = 40 ± 5 µM). The kinetics of Glu-tRNAGln and Gln-tRNAGln formation indicate that conformational shifts inside the transamidosome allow the tRNAGln acceptor stem to interact alternately with GluRS2 and GatCAB despite their common identity elements. The integrity of this dynamic transamidosome depends on a critical concentration of tRNAGln, above which it dissociates into separate GatCAB/tRNAGln and GluRS2/tRNAGln complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNAGln regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNAGln, ensuring faithful decoding of Gln codons.

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

In the cytoplasm of all eukaryotic cells and in many bacterial and archaeal species, glutamine (Gln) and asparagine (Asn) are directly esterified by discriminating aminoacyl-tRNA synthetases (D-aaRSs) on cognate transfer ribonucleic acid (tRNA) molecules, for their use in protein biosynthesis on ribosomes. By contrast, in most bacterial and archaeal species, Gln-tRNAGln and/or Asn-tRNAAsn are formed by the modification on tRNA of the side chain of their precursor amino acids glutamic acid and aspartic acid, respectively. Two enzymes participate in each of these indirect pathways: first, misacylated Glu-tRNAGln and Asp-tRNAAsn are formed by the action of nondiscriminating aminoacyl-tRNA synthetases (ND-aaRSs), ND-GluRS and ND-AspRS, respectively; then, an aminoacyl-tRNA amidotransferase (AdT) catalyzes the amidation of these misacylated tRNAs into Gln-tRNAGln and Asn-tRNAAsn, respectively. These correctly aminoacylated tRNAs are then recognized by an elongation factor to participate in ribosomal protein synthesis. High-resolution X-ray structures of these D- and ND-aaRSs and of AdT, and those of their complexes with substrates, reaction intermediates, or inhibitors, as well as kinetic and phylogenetic studies summarized in this chapter, indicate that the direct pathways catalyzed by D-GluRS and D-AspRS evolved from the more ancestral indirect pathways catalyzed by ND-GluRS, ND-AspRS, and AdT. Channeling of the misacylated Asp-tRNAAsn from ND-AspRS to AdT was discovered recently and may contribute to the fidelity of the translation of the genetic information. The ancestral character of these indirect pathways suggests that the introduction of glutamine and asparagine into proteins started later in evolution than that of their biosynthetic precursors glutamic acid and aspartic acid, as well as most other amino acids found as residues in the proteins of extant organisms. The fact that both AsnRS and Asn synthetase evolved from an ancestral AspRS by gene duplication also supports the model of a late appearance of free Asn (and of AsnRS) in the diverse community of primitive cells present on earth during the early steps of life. Gln and Asn residues are sensitive to deamidation even under physiological conditions; this sensitivity is used to control protein turnover and many other biological processes. Several efficient inhibitors of enzymes involved in the formation of Gln-tRNA and Asn-tRNA were designed and used as probes for mechanistic studies or as ligands for X-ray crystal structure determination, but selective and high whole-cell activity against microorganisms remained elusive. The availability of these enzymes for high-throughput screening provides an opportunity for developing new inhibitors. The widespread use of the transamidation pathway among bacteria and its absence in the mammalian cytoplasm identify AdT as a promising target for antibiotic therapy.

Dans cet article, l’auteur présente des arguments démontrant l’utilité d’analyser les besoins d’apprentissage ainsi que la nécessité d’intégrer cette pratique dans le développement systématique des systèmes d’enseignement. Il développe son argumentation selon les thèmes suivants : les finalités déclarées et les finalités poursuivies; la fidélité, la validité et l’utilité des finalités visées; l’incongruence des finalités par rapport aux divers niveaux de décision du système éducatif; l’expression vague des finalités poursuivies; la diversité des finalités souhaitées selon la nature des groupes consultés; la mesurabilité, critère trop souvent utilisé seul dans le choix des objectifs; la confusion entre les fins et les moyens.

Cet article propose deux aspects différents du concept de besoin en éducation : l’aspect interne et l’aspect externe. Ces deux aspects sont développés à partir de considérants relatifs aux variables en jeu dans le processus d’enseignement/apprentissage, aux critères pour en vérifier l’efficacité ainsi qu’aux éléments que l’on retrouve habituellement inclus dans la définition du mot « besoin ». De plus, on y retrouve une description, puis une comparaison des variables en jeu dans les deux types de besoins. Puis finalement, on y propose différentes décisions à prendre selon que les données générées proviennent d’une identification de besoins internes ou d’une identification de besoins externes.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. l-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with Ki values in the micromolar range (4–27μM for human mt- and mammalian cyt-AspRSs). 5′-O-[N-(l-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.

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).

La technologie de l’éducation est d’abord traitée en tant qu’outil d’intervention rationnelle orientant l’intuition du technologue dans la recherche, le développement, l’adaptation et l’application de solutions satisfaisantes aux problèmes que rencontre le praticien. Puis, elle est envisagée sous l’angle de la dynamique des systèmes à travers laquelle sont traités les intrants, les transformations et les extrants du processus. Les extrants y sont abordés selon trois aspects différents’: l’utilisation de « mini-théories », le recadrage de la situation problématique et le développement de stratégies de résolution de problèmes.

Recent genomic studies revealed the absence of glutaminyl-tRNA synthetase and/or asparaginyl-tRNA synthetase in many bacteria and archaea. The survival of microorganisms missing one or both of these essential enzymes implies an alternative pathway for the formation of Gln-tRNA(Gln) and Asn-tRNA(Asn). This indirect pathway involves misacylation of tRNA(Gln) with Glu (or tRNA(Asn) with Asp) by a nondiscriminating synthetase and subsequent transamidation of the misacylated aa-tRNA by an amidotransferase. Analogues of the natural product puromycin have been developed to inhibit this class of enzymes which has been only recently investigated at the mechanistic and structural levels. The widespread use of the indirect transamidation pathway among prominent human pathogens and its absence in the cytosol of mammalian cells identify aminoacyl-tRNA amidotransferases as an interesting target for the development of antimicrobial agents with a novel mode of action. These mechanism-based inhibitors will provide useful probes for further mechanistic investigations and ligands for X-ray crystallography.

The trimeric GatCAB aminoacyl-tRNA amidotransferases catalyze the amidation of Asp-tRNAAsn and/or Glu-tRNAGln to Asn-tRNAAsn and/or Gln-tRNAGln, respectively, in bacteria and archaea lacking an asparaginyl-tRNA synthetase and/or a glutaminyl-tRNA synthetase. The two misacylated tRNA substrates of these amidotransferases are formed by the action of nondiscriminating aspartyl-tRNA synthetases and glutamyl-tRNA synthetases. We report here that the presence of a physiological concentration of a nondiscriminating aspartyl-tRNA synthetase in the transamidation assay decreases the Km of GatCAB for Asp-tRNAAsn. These conditions, which were practical for the testing of potential inhibitors of GatCAB, also allowed us to discover and characterize two novel inhibitors, aspartycin and glutamycin. These analogues of the 3'-ends of Asp-tRNA and Glu-tRNA, respectively, are competitive inhibitors of the transamidase activity of Helicobacter pylori GatCAB with respect to Asp-tRNAAsn, with Ki values of 134 microM and 105 microM, respectively. Although the 3' end of aspartycin is similar to the 3' end of Asp-tRNAAsn, this analogue was neither phosphorylated nor transamidated by GatCAB. These novel inhibitors could be used as lead compounds for designing new types of antibiotics targeting GatCABs, since the indirect pathway for Asn-tRNAAsn or Gln-tRNAGln synthesis catalyzed by these enzymes is not present in eukaryotes and is essential for the survival of the above-mentioned bacteria.