Search

Yeast methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRS) possess N-terminal extensions that bind the cofactor Arc1p in trans. The strength of GluRS-Arc1p interaction is high enough to allow copurification of the two macromolecules in a 1:1 ratio, in contrast to MetRS. Deletion analysis from the C-terminal end of the GluRS appendix combined with previous N-terminal deletions of GluRS allows restriction of the Arc1p binding site to the 110-170 amino acid region of GluRS. This region has been shown to correspond to a novel protein-protein interaction domain present in both GluRS and Arc1p but not in MetRS [Galani, K., Grosshans, H., Deinert, K., Hurt, E. C., and Simos, G. (2001) EMBO J. 20, 6889-6898]. The GluRS apoenzyme fails to show significant kinetics of tRNA aminoacylation and charges unfractionated yeast tRNA at a level 10-fold reduced compared to Arc1p-bound GluRS. The K(m) values for tRNA(Glu) measured in the ATP-PP(i) exchange were similar for the two forms of GluRS, whereas k(cat) is increased 2-fold in the presence of Arc1p. Band-shift analysis revealed a 100-fold increase in tRNA binding affinity when Arc1p is bound to GluRS. This increase requires the RNA binding properties of the full-length Arc1p since Arc1p N domain leaves the K(d) of GluRS for tRNA unchanged. Transcripts of yeast tRNA(Glu) were poor substrates for measuring tRNA aminoacylation and could not be used to clarify whether Arc1p has a specific effect on the tRNA charging reaction.

The glutamyl-tRNA synthetase (gltX) gene from Pseudomonas aeruginosa was identified. A plasmid containing a 2.3-kb insert complemented the temperature-sensitive gltX mutation of Escherichia coli JP1449, and GltX activity was demonstrated. The inferred amino acid sequence of this gene showed 50.6% identity with GltX from Rhizobium meliloti.

The Bacillus subtilis glutamyl-tRNA synthetase (GluRS), encoded by the gltX gene, aminoacylates its homologous tRNA(Glu) and tRNA(Gln) with glutamate. This gene was cloned with its sigma A promoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 for Escherichia coli and B. subtilis. Transformation of B. subtilis with this recombinant plasmid (pMP411) led to a 30-fold increase of glutamyl-tRNA synthetase specific activity in crude extracts. Transformation of E. coli with this plasmid gave no recombinants, but transformation with plasmids bearing an altered gltX was successful. These results indicate that the presence of B. subtilis glutamyl-tRNA synthetase is lethal for E. coli, probably because this enzyme glutamylates tRNA1(Gln) in vivo as it does in vitro.

Glutamyl-tRNA synthetase (GluRS) belongs to the class I aminoacyl-tRNA synthetases and shows several similarities with glutaminyl-tRNA synthetase concerning structure and catalytic properties. Phylogenetic studies suggested that both diverged from an ancestral glutamyl-tRNA synthetase responsible for the gluta-mylation of tRNA(Glu) and tRNA(Gln), and whose Glu-tRNA(Gln) product is transformed into Gln-tRNA(Gln) by a specific amidotransferase. This pathway is present in gram-positive and some gram-negative eubacteria, in some archae and in organelles, and was never found jointly with a glutaminyl-tRNA synthetase. Other gram-negative eubacteria and the cytoplasm of eukaryotes contain a glutamyl-tRNA synthetase specific for tRNA(Glu), and a glutaminyl-tRNA synthetase. Bacterial glutamyl-tRNA synthetases consist of about 500 amino acid residues, possess molecular masses of about 50 kDa, and act as monomers. In higher eukaryotes chimeric glutamyl-prolyl-tRNA synthetases were found, in a high molecular mass complex containing several other aminoacyl-tRNA synthetases. To date one crystal structure of a glutamyl-tRNA synthetase (Thermus thermophilus) has been solved. The molecule has the form of a bent cylinder and consists of four domains. The N-terminal half (domains 1 and 2) contains the 'Rossman fold' typical for class I synthetases and resembles the corresponding part of E. coli GlnRS, whereas the C-terminal half exhibits a GluRS-specific structure. As found for the other aminoacyl-tRNA synthetases the catalytic pathway of GluRS includes the formation of an aminoacyl adenylate in the first reaction step, but GluRS shares a special property with GlnRS and ArgRS: the ATP/PPi pyrophosphate exchange reaction is only catalyzed in the presence of the cognate tRNA. Compared with other aminoacyl-tRNA synthetases a relatively high number of investigations deals with recognition of tRNA(Glu) by GluRS. Besides interactions between the enzyme and the acceptor stem and the anticodon of tRNA(Glu), checking of the dihydrouridine arm and of the variable loop by GluRS are documented.

The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc. This metal ion is strongly bound, as it is not removed by 8 M urea. Slow removal of the zinc at 4 degrees C in the presence of the specific chelating agent, 1,10-phenanthroline, is proportional to the loss of aminoacylation activity and to the presence of a more open conformer of the enzyme. This conformer migrates more slowly than the native enzyme during gel electrophoresis under nondenaturing conditions and binds tRNA(Glu). Infrared spectroscopy measurements show that it differs from the native enzyme by a lower alpha-helix content and a higher proportion of beta-sheet and unordered structures. ATP protects the enzyme against 1,10-phenanthroline-mediated zinc removal, suggesting that the zinc-binding region is closely associated with the catalytic site. Additional support for this conclusion comes from the presence of zinc in the 27-kDa N-terminal half of the enzyme and in a 10-kDa fragment. The latter is homologous to the tRNA acceptor helix binding domain of E. coli glutaminyl-tRNA synthetase. The presence of the conserved CYC motif in this domain of the zinc-containing glutamyl-tRNA synthetases of E. coli and Bacillus subtilis, and its absence in that of Thermus thermophilus and the E. coli glutaminyl-tRNA synthetase which do not contain zinc, suggest that the cysteines of this motif and the C- and H-rich 125CRHSHEHHX5C138 segment present in the 10-kDa zinc-binding fragment are involved in zinc binding by the glutamyl-tRNA synthetase of E. coli.

Rat liver prolyl-tRNA synthetase was purified as a dimer of M(r) 60,000 subunits not associated with other aminoacyl-tRNA synthetases and as a form associated with glutamyl-tRNA synthetase. Proteolysis of the dimeric enzyme generated a less active form with M(r) 52,000 subunits and an inactive form with M(r) 40,000 subunits. A second species was isolated with polypeptides of M(r) 60,000 and 150,000. This form dissociated during gel filtration chromatography being partially resolved into the M(r) 150,000 and 60,000 components; glutamyl-tRNA synthetase was associated with the larger polypeptide and prolyl-tRNA synthetase with the smaller component. Antibodies against the M(r) 60,000 polypeptide reacted with the M(r) 60,000 and 150,000 polypeptides. Gel filtration of extracts revealed multiple forms of prolyl- and glutamyl-tRNA synthetase. Antibody against the M(r) 60,000 component detected the M(r) 60,000 and 150,000 polypeptides throughout the chromatogram; these forms could be partially separated by polyethylene glycol fractionation. The M(r) 150,000 and 60,000 polypeptides were detected by Western blot analysis of crude extracts prepared under several conditions. Antibody to prolyl-tRNA synthetase reacted with a M(r) 150,000 polypeptide of the aminoacyl-tRNA synthetase core complex identified previously as glutamyl-tRNA synthetase.

In Bacillus subtilis, the glutamyl-tRNA synthetase [L-glutamate:tRNA(Glu) ligase (AMP-forming), EC 6.1.1.17] is copurified with a polypeptide of M(r) 46,000 that influences its affinity for its substrates and increases its thermostability. The gene encoding this regulatory factor was cloned with the aid of a 41-mer oligonucleotide probe corresponding to the amino acid sequence of an NH2-terminal segment of this factor. The nucleotide sequence of this gene and the physical map of the 1475-base-pair fragment on which it was cloned are identical to those of purB, which encodes the adenylosuccinate lyase (adenylosuccinate AMP-lyase, EC 4.3.2.2), an enzyme involved in the de novo synthesis of purines. This gene complements the purB mutation of Escherichia coli JK268, and its presence on a multicopy plasmid behind the trc promoter in the purB- strain gives an adenylosuccinate lyase level comparable to that in wild-type B. subtilis. A complex between the adenylosuccinate lyase and the glutamyl-tRNA synthetase was detected by centrifugation on a density gradient. The interaction between these enzymes may play a role in the coordination of purine metabolism and protein biosynthesis.

The glutamyl-tRNA synthetase (EC 6.1.1.17) of Escherichia coli was purified to homogeneity from the overproducing strain DH5 alpha(pLQ7612) by a two-step procedure that takes only about 6 h and yields 10 mg of enzyme per gram of wet cells. The process consists of a two-phase polyethylene glycol-dextran partition, the top phase of which is diluted and directly applied to an anion-exchange FPLC MonoQ column. The purified enzyme has a specific activity about twice that of the same enzyme purified to homogeneity by the lengthy conventional procedure from either a normal strain or this overproducing strain. This difference is discussed in relation to the generation of microheterogeneity in proteins during their purification.

Formation of the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate via the five-carbon pathway requires three enzymes: glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde (GSA) aminotransferase. All three enzymes were separated from extracts of the unicellular cyanobacterium Synechocystis sp. PCC 6803, and two of them, glutamyl-tRNA synthetase and GSA aminotransferase, were partially characterized. After an initial high speed centrifugation and differentiatial ammonium sulfate fractionation of cell extract, the enzymes were separated by successive affinity chromatography on Reactive Blue 2-Sepharose and 2',5'-ADP-agarose. All three enzyme fractions were required to reconstitute ALA formation from glutamate. The apparent native molecular masses of glutamyl-tRNA synthetase and GSA aminotransferase were determined by gel filtration chromatography to be 63 and 98 kDa, respectively. Neither glutamyl-tRNA synthetase nor GSA aminotransferase activity was affected by hemin concentrations up to 10 and 30 microM, respectively, and neither activity was affected by protochlorophyllide concentrations up to 2 microM. GSA aminotransferase was inhibited 50% by 0.5 microM gabaculine. The gabaculine inhibition was reversible for up to 1 h after its addition, if the gabaculine was removed by gel filtration before the enzyme was incubated with substrate. However, irreversible inactivation was obtained by preincubating the enzyme at 30 degrees C either for several hours with gabaculine alone or for a few minutes with both gabaculine and GSA. Neither pyridoxal phosphate nor pyridoxamine phosphate significantly affected the activity of GSA aminotransferase at physiologically relevant concentrations, and neither of these compounds reactivated the gabaculine-inactivated enzyme. It was noted that the presence of pyridoxamine phosphate in the ALA assay mixture produced a false positive color reaction even in the absence of enzyme.

The formation of delta-aminolevulinic acid, the first committed precursor in porphyrin biosynthesis, occurs in certain bacteria and in the chloroplasts of plants and algae in a three-step, tRNA-dependent transformation of glutamate. Glutamyl-tRNA reductase, the second enzyme of this pathway, reduces the activated carboxyl group of glutamyl-tRNA (Glu-tRNA) in the presence of NADPH and releases glutamate 1-semialdehyde (GSA). We have purified Glu-tRNA reductase from Chlamydomonas reinhardtii by employing six different chromatographic separations. The apparent molecular mass of the protein when analyzed under both denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 130,000 Da; this indicates that the active enzyme is a monomer. In the presence of NADPH Glu-tRNA reductase catalyzed the reduction to GSA of glutamate acylated to the homologous tRNA. Thus, the reductase alone is sufficient for conversion of Glu-tRNA to GSA. In the absence of NADPH, a stable complex of Glu-tRNA reductase with Glu-tRNA can be isolated.

Glutamyl-tRNA synthetase from Chlamydomonas reinhardtii was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, Mono Q, and Mono S. The apparent molecular mass of the protein when analyzed under both denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 62,000 Da; this indicates that the active enzyme is a monomer. The purified glutamyl-tRNA synthetase was identified as the chloroplast enzyme by its tRNA charging specificity. Reversed-phase chromatography of unfractionated C. reinhardtii tRNA resolved four peaks of glutamate acceptor RNA when assayed with the purified enzyme. The enzyme can also glutamylate Escherichia coli tRNA(2Glu), but not cytoplasmic tRNA(Glu) from yeast or barley. In addition, the enzyme misacylates chloroplast tRNA(Gln) with glutamate. A similar mischarging phenomenon has been demonstrated for the barley chloroplast enzyme (Schön, A., Kannangara, C.G., Gough, S., and Söll, D. (1988) Nature 331, 187-190) and for Bacillus subtilis glutamyl-tRNA synthetase (Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M., and Lapointe, J. (1983) J. Biol. Chem. 258, 753-759).

Three dimeric glutamyl-tRNA synthetases (GluRS) were isolated from extracts of quiescent wheat germ and wheat chloroplasts. The chloroplast enzyme (Mr = 110 000), called GluRS C, exhibits a prokaryotic (Escherichia coli) tRNA specificity. Two enzymes were found in the quiescent germ and were separated on phosphocellulose P11: one called GluRS P, probably the mitochondrial enzyme, has the same tRNA specificity as GluRS C; the other, called GluRS E, has eukaryotic (wheat germ) tRNA specificity. Both enzymes exhibit a molecular weight close to 160 000. Each of these enzymes co-eluate on hydroxyapatite and phosphocellulose chromatographies with an unstable active monomer whose molecular weight is approximately half that of the corresponding dimer. Two assumptions are discussed about these monomers.

The glutamyl-tRNA synthetase purified 300-fold from calf brain is associated with other aminoacyl-tRNA synthetases in a complex whose molecular weight is about 2,000,000. However, in a less purified state, the enzyme is present in a complex larger than 5,000,000. The properties of the enzyme are the same in both complexes except for the pH optimum of the aminoacylation reaction. The presence of 2-mercaptoethanol protects and increases the enzymatic activity. gamma-Methyl-L-glutamate and salicylate show competitive inhibition with respect to glutamate but kainic acid and taurine have no effect on the rate of aminoacylation of tRNAGlu.

The interaction of glutamyl-tRNA synthetase with tRNAGlu2 has been studied. The enzyme was purified to apparent homogeneity, and consists of a single chain with a molecular weight of 59 000. The sedimentation coefficient (sdegrees20,w) was found to be 3.7 S and suggests this enzyme is quite asymmetric. The enzyme binds 1 mol of tRNAGlu2 and has a binding constant of 5 X 10(6) M-1 at pH 7.0 in 0.1 M sodium chloride. A circular dichroic study of the interaction under the same solvent conditions implied both the synthetase and tRNAGlu2 underwent a change in conformation as the complex was formed. In the case of the enzyme there appears to be some loss of alpha-helical structure. The tRNAGlu2 results can be interpreted to indicate a change in the conformation of one or more of the helical regions of this molecule. A residue in the anticodon loop, 5-methylaminomethyl-2-thiouridine, has a distinct circular dichroic band at 340 nm in the free tRNAGlu2. As the complex is formed this band is shifted to the blue. This was interpreted to indicate that the enzyme forms a hydrogen bond with this residue in the anticodon loop, with a change in the conformation of the loop possibly also having occured.

The gltX gene, coding for the glutamyl-tRNA synthetase of Rhizobium meliloti A2, was cloned by using as probe a synthetic oligonucleotide corresponding to the amino acid sequence of a segment of the glutamyl-tRNA synthetase. The codons chosen for this 42-mer were those most frequently used in a set of R. meliloti genes. DNA sequence analysis revealed an open reading frame of 484 codons, encoding a polypeptide of Mr 54,166 containing the amino acid sequences of an NH2-terminal and various internal fragments of the enzyme. Compared with the amino acid sequence of the glutamyl-tRNA synthetase of Escherichia coli, the N-terminal third of the R. meliloti enzyme was strongly conserved (52% identity); the second third was moderately conserved (38% identity) and included a few highly conserved segments, whereas no significant similarity was found in the C-terminal third. These results suggest that the C-terminal part of the protein is probably not involved in the recognition of substrates, a feature shared with other aminoacyl-tRNA synthetases.

The glutamyl-tRNA synthetase has been purified to homogeneity from Escherichia coli with a yield of about 50%. It is a monomer with a molecular weight of 56,000 and has the same kinetic properties as those of the alpha chain of the dimeric alphabeta-glutamyl-tRNA synthetase described previously (Lapointe, J., and Söll, D. (1972) J. Biol. Chem. 247, 4966-4974). It is the smallest amino-acyl-tRNA synthetase purified from E. coli and contains no important sequence repetition. It is also the only monomeric aminoacyl-tRNA synthetase reported so far to contain no major sequence duplication. Considering its structural and mechanistic similarities with the glutaminyl- and the arginyl-tRNA synthetases of E. coli, we propose the existence of a relation between the true monomeric character of the glutamyl-tRNA synthetase (as opposed to monomers with sequence duplications) and its requirement for tRNA in the activation of glutamate. A single sulfhydryl group of the native enzyme reacts with 5,5'-dithiobis(2-nitrobenzoic acid) causing no loss of enzymatic activity, whereas four such groups per enzyme react in the presence of 4 M guanidine HCl.

The Michaelis constants in the tRNA aminoacylation reaction have been studied for the three dimeric glutamyl-tRNA synthetases C, P and E. The values were found to be: for tRNA, 0.20 microM, and 0.44 microM; for glutamic acid, 10 microM, 83 microM and 83 microM; for MgATP, 0.46 mM, 0.38 mM and 0.26 mM. MgATP concentrations higher than 2 mM induce pronounced inhibition. The presence of the cognate tRNA is required for [32P]PPi-ATP isotopic exchange. In the absence of tRNA no hyperbolic saturation of the enzymes by glutamic acid occurs in our experimental conditions. Analysis of the enzymic activity as a function of enzyme concentration leads to the conclusion that the active forms are dimers which are in equilibrium with inactive monomers. The values of the dissociation constants Kd were found to be 43 nM, 53 nM and 87 nM for glutamyl-tRNA synthetases C, P and E respectively.

A glutamyl-tRNA synthetase has been purified to homogeneity from Rhizobium meliloti, using reversed-phase chromatography as the last step. Amino acid sequencing of the amino-terminal region of the enzyme indicates that it contains a single polypeptide, whose molecular weight is about 54,000, as judged by SDS-gel electrophoresis. The primary structures of the amino-terminus region and of an internal peptide obtained by cleavage of the enzyme with CNBr have similarities of 58 and 48% with regions of the glutamyl-tRNA synthase of Escherichia coli; these are thought to be involved in the binding of ATP and tRNA, respectively. The small amount of glutamyl-tRNA synthetase present in R. meliloti is consistent with the metabolic regulation of the biosynthesis of many aminoacyl-tRNA synthetases.

Glutamyl-tRNA synthetase has been isolated from an extreme thermophile, Thermus thermophilus HB8. The enzyme has been purified to homogeneity by successive chromatography on columns of DEAE-cellulose, DEAE-Sephacel, phosphocellulose and hydroxyapatite. 11.7 mg of purified enzyme has been obtained from 2 kg of T. thermophilus cells, with a purification factor of 600 with an 11% yield. From gel permeation chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme is found to be a monomer protein with a molecular weight of 50,000. The optimum temperature for the aminoacylation of T. thermophilus tRNAGlu is 65 degrees C, and the optimum pH range is 8.0-9.0, in the presence of 5 mM Mg2+. The Km values for ATP, L-glutamate, and T. thermophilus tRNAGlu are 230 microM, 70 microM, and 0.65 microM, respectively, in the presence of 50 mM KCl and 10 mM MgCl2 at pH 8.0 at 65 degrees C. Escherichia coli tRNA2Glu is also a good substrate with a Km value of 0.60 microM at 65 degrees C. The mole fractions of Arg and Leu residues are higher and that of Asx residues is lower than those of E. coli glutamyl-tRNA synthetase. Glutamyl-tRNA synthetase from T. thermophilus is remarkably thermostable; even after incubation for 9 h at 65 degrees C, 70% of the enzyme activity is retained in the absence of any protecting factors. Such an extremely thermostable enzyme with a low molecular weight will be useful for detailed physiochemical analyses on the molecular mechanism of strict recognition by aminoacyl-tRNA synthetases.