Purification of isoleucyl-tRNA synthetase from Methanobacterium thermoautotrophicum by pseudomonic acid affinity chromatography

The isoleucyl-tRNA synthetase of the archaebacterium Methanobacterium thermoautotrophicum was purified 1500-fold to electrophoretic homogeneity by a procedure based on affinity chromatography on Sepharose-bound pseudomonic acid, a strong competitive inhibitor of this enzyme. The purified enzyme is a monomer with a molecular mass of 120 kDa. In this respect and in its Km values for the PPi-ATP exchange, and aminoacylation reactions, it resembles the isoleucyl-tRNA synthetases from eubacterial and eukaryotic sources. Its aminoacylation activity is optimal at pH 8.0 and at 55 degrees C. Pseudomonic acid is a strong competitive inhibitor of the aminoacylation reaction with respect to both L-isoleucine (KiIle 10 nM) and ATP (KiATP 20 nM).     

Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.     

Interacting binding sites of isoleucyl-tRNA synthetase from Escherichia coli studied by equilibrium partition.

The binding of tRNAIIe to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIIe for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine. A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.     

The valyl-tRNA synthetase from Bacillus stearothermophilus has considerable sequence homology with the isoleucyl-tRNA synthetase from Escherichia coli

We report the DNA sequence of the valS gene from Bacillus stearothermophilus and the predicted amino acid sequence of the valyl-tRNA synthetase encoded by the gene. The predicted primary structure is for a protein of 880 amino acids with a molecular mass of 102,036. The molecular mass and amino acid composition of the expressed enzyme are in close agreement with those values deduced from the DNA sequence. Comparison of the predicted protein sequence with known protein sequences revealed a considerable homology with the isoleucyl-tRNA synthetase of Escherichia coli. The two enzymes are identical in some 20-25% of their amino acid residues, and the homology is distributed approximately evenly from N-terminus to C-terminus. There are several regions which are highly conservative between the valyl- and isoleucyl-tRNA synthetases. In one of these regions, 15 of 20 amino acids are identical, and in another, 10 of 14 are identical. The valyl-tRNA synthetase also contains a region HLGH (His-Leu-Gly-His) near its N-terminus equivalent to the consensus HIGH (His-Ile-Gly-His) sequence known to participate in the binding of ATP in the tyrosyl-tRNA synthetase. This is the first example of extensive homology found between two different aminoacyl-tRNA synthetases.     

On the binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

The binding of nine aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 was measured and compared with the binding of the cognate amino acids. It was found that they bind rather tightly to the enzyme, the Kd's ranging from 3.1.10(-4) M with glycinol-AMP ester to 3.7.10(-9) M with L-isoleucinol-AMP ester. The binding is not affected by magnesium. It is shown that the free energies of binding of the esters can be calculated adding a constant contribution of the AMP-moiety of about - 4.1 (- 17) kcal/mole (kJ/mole) to the free energies of binding of the cognate amino acids, which we have reported earlier (19, 25, 26).     

Evidence for cAMP-mediated control of isoleucyl-tRNA synthetase formation in Escherichia coli K-12

The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl-tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP.Abbreviations IRS isoleucyl-tRNA synthetase - VRS valyl-tRNA synthetase - ARS arginyl-tRNA synthetase - cAMP cyclic adenosine-3,5-monophosphate - Cya adenyl cyclase Gene - CRP cAMP receptor protein - O.D. optical density     

Structure of the yeast valyl-tRNA synthetase gene (VASI) and the homology of its translated amino acid sequence with Escherichia coli isoleucyl-tRNA synthetase

The VASI gene encoding the valyl-tRNA synthetase from yeast was isolated and sequenced. The gene-derived amino acid sequence of yeast valyl-tRNA synthetase was found to be 23% homologous to the Escherichia coli isoleucyl-tRNA synthetase. This is the highest level of homology reported so far between two distinct aminoacyl-tRNA synthetases and is indicative of an evolutionary relationship between these two molecules. Within these homologous sequences, two functional regions could be recognized: the HIGH region which forms part of the binding site of ATP and the KMSKS region which is recognized as the consensus sequence for the binding of the 3'-end of tRNA (Hountondji, C., Dessen, Ph., and Blanquet, S. (1986) Biochemie (Paris) 68, 1071-1078). Secondary structure predictions as well as the presence of both HIGH and KMSKS regions, delineating the nucleotide-binding domain and the COOH-terminal helical domain in aminoacyl-tRNA synthetases of known three-dimensional structure, suggest that the yeast valyl-tRNA synthetase polypeptide chain can be folded into three domains: an NH2-terminal alpha-helical region followed by a nucleotide-binding topology and a COOH-terminal domain composed of alpha-helices which probably carries major sites in tRNA binding.     

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl-tRNA synthetase from Escherichia coli.

A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E.aa-AMP possible. If the E.Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E.Ile-AMP with E.Ile and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E.Ile-AMP and the corrected (*E)tRNA(Ile-AMP). If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be beneficial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.     

ATP-induced activation of the aminoacylation of tRNA by the isoleucyl-tRNA synthetase from Escherichia coli

The rate of aminoacylation of tRNA catalyzed by the isoleucyl-tRNA synthetase form Escherichia coli has been measured. A steady-state kinetic analysis of the rate as a function of the concentration of ATP gave nonlinear Hanes plots. ATP behaves as an activator of the reaction. The activation is observed at a low magnesium ion concentration and in the presence of spermidine. The presence of inorganic pyrophosphate or AMP enhances the activation. The results are consistent with a mechanism in which the binding of a second molecule of ATP increases the rate of dissociation of Ile-tRNA from the enzyme.     

Repression by glucose of acetohydroxy acid synthetase in Escherichia coli B

Acetolactate formation in Escherichia coli B results from the activity of a single system, acetohydroxy acid synthetase, which has a pH optimum of 8.0 and is sensitive to end-product inhibition by l-valine. Acetohydroxy acid synthetase was found to be subject to catabolite repression, and the nature and concentration of the carbon source had a greater effect on the formation of the enzyme than had the known end products (valine, isoleucine, leucine and pantothenate) of the biosynthetic pathways of which this enzyme is a member. The results suggest that acetohydroxy acid synthetase may play an amphibolic role in E. coli B.     

NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase

The 400-MHz 1H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS. The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche+ form about the C alpha-C beta bond and the trans form about the C beta-C gamma 1 bond. The IleRS-bound L-valine takes the gauche- form about the C alpha-C beta bond. Thus, the conformation of IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.     

Effect of polyamines on isoleucyl-tRNA formation by rat-liver isoleucyl-tRNA synthetase.

The effect of polyamines on rat-liver isoelucyl-tRNA formation was studied using isoleucyl-tRNA synthetase purified by column chromatography successively on Sephadex G-200, DEAE-Sephadex A-25, and tRNA-Sepharose 4B. In the presence of 50 mMK+, isoleucyl-tRNA formation was inhibited markedly by 1.5 mM or higher concentrations of Mg2+. However, the addition of spermine to the reaction mixture prevented the inhibitory effect of Mg2+. In the presence of 200 mMK+, the addition of spermine to the reaction mixture stimulated isoleucyl-tRNA formation in the presence of Mg2+ concentrations from 0 to 5 mM. Although the effective concentration was different, spermidine exhibited a similar stimulative effect. The effective concentration of spermine required for stimulation was higher when larger amounts of tRNA were used. The stimulatory effect of isoleucyl-tRNA formation by polyamines was shown to reflect on polypeptide synthesis. When formaldehyde-treated poly(A,U) was used as messenger RNA, polypeptide synthesis from amino acids was stimulated by polyamines, but that from aminoacyl-tRNAs was not stimulated by polyamines.     

Control of acetohydroxy acid synthetase in Escherichia coli 9723.

A method by which three acetohydroxy acid synthetase activities are separated from extracts of Escherichia coli 9723 has been developed. Isoleucine specifically represses synthesis of one of the enzymes, which is not sensitive to valine inhibition, and isoleucine also simultaneously enhances the production of a second activity, which is valine inhibitable. The valine-inhibitable activity is repressed by leucine and valine, a combination of which is more effective than either alone. The third acetohydroxy acid synthetase, which is more active at pH 6 than at 8, is not controlled by the branched-chain amino acids. In a mutant of E. coli 9723 selected for the ability of valine to inhibit growth, the isoleucine-repressible acetohydroxy acid synthetase activity was no longer present, but isoleucine addition still resulted in enhanced production of the valine-inhibitable activity.