The glutamyl-tRNA synthetase of Escherichia coli: substrate-induced protection against its thermal inactivation.

The substrates-induced protection against the heat-inactivation of the glutamyl-tRNA synthetase has been investigated. tRNAGlu and ATP protect efficiently the enzyme, whereas glutamate does not. In the presence of tRNAGlu, glutamate induces an additional protection to that given by the tRNAGlu alone. A weak synergism was observed between ATP and tRNAGlu, whereas no synergism was detected between ATP and glutamate. These results suggest that tRNAGlu and ATP, but not glutamate are able to bind to the free enzyme form; glutamate binds only to the Enzyme.tRNAGlu and to the Enzyme.tRNAGlu.ATP complexes. The presence of the three substrates induces a higher stabilization of the enzyme than that expected from the protection observed for the various other substrates combinations, suggesting the existence of a marked synergism between the three substrates against the heat-inactivation of the enzyme. The protection constants determined from this study are similar to the dissociation constants determined by direct binding experiments and to the Km values determined kinetically.     

A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln in Bacillus subtilis and efficiently misacylates Escherichia coli tRNAGln1 in vitro.

In the presence or absence of its regulatory factor, the monomeric glutamyl-tRNA synthetase from Bacillus subtilis can aminoacylate in vitro with glutamate both tRNAGlu and tRNAGln from B. subtilis and tRNAGln1 but not tRNAGln2 or tRNAGlu from Escherichia coli. The Km and Vmax values of the enzyme for its substrates in these homologous or heterologous aminoacylation reactions are very similar. This enzyme is the only aminoacyl-tRNA synthetase reported to aminoacylate with normal kinetic parameters two tRNA species coding for different amino acids and to misacylate at a high rate a heterologous tRNA under normal aminoacylation conditions. The exceptional lack of specificity of this enzyme for its tRNAGlu and tRNAGln substrates, together with structural and catalytic peculiarities shared with the E. coli glutamyl- and glutaminyl-tRNA synthetases, suggests the existence of a close evolutionary linkage between the aminoacyl-tRNA synthetases specific for glutamate and those specific for glutamine. A comparison of the primary structures of the three tRNAs efficiently charged by the B. subtilis glutamyl-tRNA synthetase with those of E. coli tRNAGlu and tRNAGln2 suggests that this enzyme interacts with the G64-C50 or G64-U50 in the T psi stem of its tRNA substrates.     

Effect of modified nucleotides on Escherichia coli tRNAGlu structure and on its aminoacylation by glutamyl-tRNA synthetase. Predominant and distinct roles of the mnm5 and s2 modifications of U34.

Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAGlu modivariant in wild-type E. coli contains five modified nucleosides: Psi13, mnm5s2U34, m2A37, T54 and Psi55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D- and T-arms and in the variable region. The lack of a 2-thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520-fold the specificity of E. coli glutamyl-tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAGlu. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl-tRNA synthetase for the recognition of glutamate and ATP.     

The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm.

In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end. We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6-25), and predicted to interact with DNA or proteins. In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively. On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS. In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm. Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain. Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site.     

Derepressed levels of glutamate synthase and glutamine synthetase in Escherichia coli mutants altered in glutamyl-transfer ribonucleic acid synthetase.

The levels of glutamate synthase and of glutamine synthetase are both derepressed 10-fold in strain JP1449 of Escherichia coli carrying a thermosensitive mutation in the glutamyl-transfer ribonucleic acid (tRNA) synthetase and growing exponentially but at a reduced rate at a partially restrictive temperature, compared with the levels in strain AB347 isogenic with strain JP1449 except for this thermosensitive mutation and the marker aro. These two enzymes catalyze one of the two pathways for glutamate biosynthesis in E. coli, the other being defined by the glutamate dehydrogenase. We observed a correlation between the percentage of charged tRNAGlu and the level of glutamate synthase in various mutants reported to have an altered glutamyl-tRNA synthetase activity. These results suggest that a glutamyl-tRNA might be involved in the repression of the biosynthesis of the glutamate synthase and of the glutamine synthetase and would couple the regulation of the biosynthesis of these two enzymes, which can work in tandem to synthesize glutamate when the ammonia concentration is low in E. coli but whose structural genes are quite distant from each other. No derepression of the level of the glutamate dehydrogenase was observed in mutant strain JP1449 under the conditions where the levels of the glutamine synthetase and of the glutamate synthase were derepressed. This result indicates that the two pathways for glutamate biosynthesis in E. coli are under different regulatory controls. The glutamate has been reported to be probably the key regulatory element of the biosynthesis of the glutamate dehydrogenase. Our results indicate that the cell has chosen the level of glutamyl-tRNA as a more sensitive probe to regulate the biosynthesis of the enzymes of the other pathway, which must be energized at a low ammonia concentration.     

Complex formation between glutamyl-tRNA synthetase and glutamyl-tRNA reductase during the tRNA-dependent synthesis of 5-aminolevulinic acid in Chlamydomonas reinhardtii.

The formation of a stable complex between glutamyl-tRNA synthetase and the first enzyme of chlorophyll biosynthesis glutamyl-tRNA reductase was investigated in the green alga Chlamydomonas reinhardtii. Apparently homogenous enzymes, purified after previously established purification protocols were incubated in various combinations with ATP, glutamate, tRNA(Glu) and NADPH and formed complexes were isolated via glycerol gradient centrifugation. Stable complexes were detected only after the preincubation of glutamyl-tRNA synthetase, glutamyl-tRNA reductase with either glutamyl-tRNA or free tRNA(Glu), ATP and glutamate, indicating the obligatory requirement of aminoacylated tRNA(Glu) for complex formation. The further addition of NADPH resulting in the reduction of the tRNA-bound glutamate to glutamate 1-semialdehyde led to the dissociation of the complex. Once complexed to the two enzymes tRNA(Glu) was found to be partially protected from ribonuclease digestion. Escherichia coli, Bacillus subtilis and Synechocystis 6803 tRNA(Glu) were efficiently incorporated into the protein-RNA complex. The detected complexes provide the chloroplast with a potential channeling mechanism for Glu-tRNA(Glu) into chlorophyll synthesis in order to compete with the chloroplastic protein synthesis machinery.     

Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants

The 2.5 A crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft. In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain. The nucleophilic alpha-carboxylate moiety of glutamate is mispositioned with respect to both the ATP alpha-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction. Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH(2) group of the cognate glutamine substrate. At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule. These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested. Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation. The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases.     

Conformation change of tRNAGlu in the complex with glutamyl-tRNA synthetase is required for the specific binding of L-glutamate

The binding of Thermus thermophilus glutamyl-tRNA synthetase (GluRS) with T. thermophilus tRNAGlu, Escherichia coli tRNAGlu, and amino acids was studied by fluorescence measurements. In the absence of tRNAGlu, GluRS binds with D-glutamate as well as L-glutamate. However, in the presence of E. coli tRNAGlu, GluRS binds specifically with L-glutamate. The KCl effects on the Michaelis constants (Km) for tRNAGlu, L-glutamate, and ATP were studied for the aminoacylation of the homologous tRNAGlu and heterologous tRNAGlu species. As the KCl concentration is raised from 0 to 100 mM, the Km value for L-glutamate in the heterologous system is remarkably increased whereas the Km value for L-glutamate in the homologous system is only slightly increased. The circular dichroism analyses were made mainly of the bands due to the 2-thiouridine derivatives of tRNAGlu in the complex. The conformation change of T. thermophilus tRNAGlu upon complex formation with GluRS is not affected by addition of KCl. In contrast, the heterologous tRNAGlu X GluRS complex is in an equilibrium of two forms that depends on KCl concentration. The predominant form at low KCl concentration is closely related to the small Km value for L-glutamate. In this form of the complex, the conformation of tRNAGlu is appreciably different from that of free molecule. Accordingly, such a conformation change of tRNAGlu in the complex with GluRS is required for the specific binding of L-glutamate as the substrate.     

The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor. Their purification, characterization, and the study of their interaction

The glutamyl-tRNA synthetase from Bacillus subtilis has been purified to homogeneity. It is a monomer of Mr = 65,500 whose NH2-terminal sequence is Met-Asn-Glu-Val-Arg-Val-Arg-Tyr-Ser-Pro-Ser-Pro-Thr-Gly-His-Leu. The number of tryptic peptides indicates the absence of a significant amount of sequence duplication. Under certain conditions, this monomeric enzyme is co-purified with a polypeptide beta of Mr = 46,000, which increases the affinity of the enzyme about 10-fold for glutamate and for ATP, and stabilizes it against heat inactivation. gamma-Globulins prepared against the monomeric enzyme can inhibit completely the glutamyl-tRNA synthetase activity of a B. subtilis extract and precipitate from this extract both the monomeric enzyme and the regulatory factor beta. These anti-alpha immunoglobulins do nt precipitate pure beta. These results show that the glutamyl-tRNA synthetase of B. subtilis has a structure similar to that of the Escherichia coli enzyme (Lapointe, J., and S?ll, D. (1972) J. Biol. Chem. 247, 4966-4974) and indicate that the beta factor has a function in the regulation of glutamyl-tRNA biosynthesis in vivo.     

Histidyl transfer ribonucleic acid synthetase from Salmonella typhimurium. Interaction with substrates and ATP analogues.

Structural requirements for substrate binding to histidyl-tRNA synthetase from Salmonella typhimurium have been investigated using ATP analogues. Ki values and the relative binding affinity of the enzyme for these analogues have been determined in the tRNA aminoacylation reaction. The enzyme is highly specific for ATP: no binding was found for GTP, CTP, TTP and UTP. dATP is a very poor substrate for acylation of tRNA, with a Km 40-fold higher than that of ATP. Binding of adenosine 5'-triphosphate requires interactions of the amino group of adenosine and the sugar moiety; the 2' and the 5' positions of the ribose appear to be essential for recognition; the phosphate groups enhance the binding. AMP is a noncompetitive inhibitor with ATP. The interaction of histidyl-tRNA synthetase, a dimeric enzyme, with histidine and ATP was examined by fluorescence measurements at equilibrium and by equilibrium dialysis. Binding with L-histidine is significantly tighter at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 6 than at pH 7, while the ATP binding is independent of pH. The stoichiometry was measured at pH 7.5 by equilibrium dialysis and is 1 mol ATP/mol enzyme and, variably, close to 2 or 1 mol histidine/mol enzyme.     

Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme.

The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.     

Identification of the enzymatic basis for delta-aminolevulinic acid auxotrophy in a hemA mutant of Escherichia coli.

The hemA mutation of Escherichia coli K-12 confers a requirement for delta-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem+ strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-[14C]glutamate and 3,4-[3H]glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNAGlu. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem+ cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem+ cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.     

The conformations of a substrate and a product bound to the active site of S-adenosylmethionine synthetase

S-Adenosylmethionine (AdoMet) is the most widely used alkyl group donor in biological systems. The formation of AdoMet from ATP and L-methionine is catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase). Elucidation of the conformations of enzyme-bound substrates, product, and inhibitors is important for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. To obtain structural data for enzyme-bound substrates and product, we have used two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzyme-bound AdoMet and 5'-adenylyl imidodiphosphate (AMPPNP). AMPPNP, an analogue of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a nonhydrolyzable NH-link between the beta- and gamma-phosphates but is a substrate for AdoMet formation during which tripolyphosphate is produced. AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond. The ribose rings are in C3'-exo and C4'-exo conformations in AdoMet and AMPPNP, respectively. The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet. The NMR-determined conformations of AdoMet and AMPPNP were docked into the E. coli AdoMet synthetase active site taken from the enzyme.ADP. Pi crystal structure. Since there are no nonexchangeable protons either in the carboxy-terminal end of the methionine segment of AdoMet or in the tripolyphosphate segment of AMPPNP, these portions of the molecules were modeled into the enzyme active site. The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and suggest how the positive charge formed on the sulfur during AdoMet synthesis is stabilized.     

Mechanistic investigations of Escherichia coli cytidine-5'-triphosphate synthetase. Detection of an intermediate by positional isotope exchange experiments

CTP synthetase from Escherichia coli catalyzes exchange of 18O from the beta gamma-bridge position of [gamma-18O4] ATP into the beta-nonbridge position. This positional isotope exchange occurs in the presence of UTP and MgCl2 but in the absence of NH3. The enzyme also has an ATPase activity in the presence of UTP that occurs under conditions that are identical to those used in the positional isotope exchange experiments. These data provide evidence for the stepwise nature of the reactions catalyzed by CTP synthetase with the initial step involving phosphorylation of UTP by ATP. The relative rate of the isotope exchange reaction is approximately 3 times faster than the ATPase reaction, but the isotope exchange rate is approximately 3% of the overall rate in the presence of NH3. These results are consistent with the ATPase reaction involving attack of water on the phosphorylated intermediate (4-phospho-UTP). The positional isotope exchange reaction is independent of the UTP concentration above saturating levels of UTP demonstrating that the order of addition of substrates is UTP followed by ATP and then NH3.     

Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli

The charging of glutamate on tRNA(Glu) is catalyzed by glutamyl-tRNA synthetase, a monomer of 53.8 kilodaltons in Escherichia coli. To obtain the large amounts of enzyme necessary for the identification of structural domains, we have inserted the structural gene gltX in the conditional runaway-replication plasmid pOU61, which led to a 350-fold overproduction of glutamyl-tRNA synthetase. Partial proteolysis of this enzyme revealed the existence of preferential sites of attack that, according to their N-terminal sequences, delimit regions of 12.9, 2.3, 12.1, and 26.5 kilodaltons from the N- to C-terminal of the enzyme. Their sizes suggest that the 2.3-kilodalton fragment is a hinge structure, and that those of 12.9, 12.1, and 26.5 kilodaltons are domain structures. The 12.9-kilodalton domain of the glutamyl-tRNA synthetase of E. coli is the only long region of this enzyme displaying a good amino acid sequence similarity with the glutaminyl-tRNA synthetase of Escherichia coli.     

ATP-analogues as substrates for the leucyl-tRNA synthetase from Escherichia coli MRE 600.

No analogous nucleoside triphosphate was found which acts as well as ATP in binding to and supporting catalysis of leucyl-tRNA synthetase from Escherichia coli MRE 600. However, there are numerous nucleotides which are able to replace ATP, but with lower efficiency. The 6-amino group of the adenine ring and the 2'-hydroxyl group of the ribose ring are essential for binding and catalytic activity. Alterations in the triphosphate moiety of the molecule can cause drastic changes in Km and/or Vmax, whereas alterations of the imidazole ring and substitutions at the 8-position of the adenine ring cause only minor losses of catalytic activity.     

Ester and hydroxamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases

The structure activity relationship on a series of ester and hydroxamate analogues of methionyl and isoleucyl adenylate has been investigated through introducing linkers between the 1'-position of ribose and adenine surrogates as methionyl-tRNA, and isoleucyl-tRNA synthetase inhibitors, respectively. The results indicate that ester analogue 23 was found to be a potent inhibitor of Escherichia coli methionyl-tRNA synthetase, and its interaction with the active site was proposed by a molecular modeling study.     

Binding of tyrosine, adenosine triphosphate and analogues to crystalline tyrosyl transfer RNA synthetase

Crystalline complexes of tyrosyl tRNA synthetase were prepared with the following substrates and substrate analogues: ATP, AMP, α-β methylene ATP, tyrosine and tyrosinyl adenylate. Using ¹⁴C-labelled ligands, the binding constants for tyrosine and ATP to crystals were shown to be similar to those observed in solution. Two tyrosine molecules were found to bind to the symmetrical dimer in the crystalline enzyme, while only one tyrosine binds with high affinity in solution. Electron density difference maps show that tyrosine and the AMP derivatives all bind at the same site, in a cleft 10 Å deep at one side of the pleated sheet, tyrosine binding over 100 times more strongly. The phosphate groups of AMP and ATP are not unambiguously observed in the difference electron density maps. Tyrosinyl adenylate is clearly delineated in the electron density difference map, with the tyrosyl side-chain occupying the site previously observed. The adenosine group is in a wide cup-like depression outside the pocket, lying between the carboxyl-terminal continuations of strands 3 and 5 of the pleated sheet. The adenine ring is lying against an α-helix. The binding of tyrosinyl adenylate causes no detectable conformational changes of the enzyme.     

A stereochemical method for detection of ATP terminal phosphate transfer in enzymatic reactions. Glutamine synthetase.

An isotope scrambling method is described for the detection of transient [Enz:ADP:P-X] formation from [18O]ATP in ATP-coupled enzyme reactions. The method makes use of torsional symmetry of the newly formed (see article) group in ADP. [18 O]ATP labeled in the betagama bridge oxygen was incubated with enzyme and reversible cleavage of the PbetaO -- Pgamma bond was detected by the appearance of 18O in the beta nonbridge oxygens of the ATP pool. Experiments with sheep brain and Escherichia coli glutamine synthetases show that cleavage of ATP of enzyme-bound ADP and P-X requires glutamate. The exchange catalyzed by the E. coli enzyme with glutamate occurs in the absence of ammonia and is partially inhibited by added NH4Cl, as expected if the exchange is in the mechanistic pathway for glutamine synthesis. The results provide kinetic support for a two-step mechanism where phosphoryl transfer from ATP to glutamate precedes reaction with ammonia.