Valyl-tRNA synthetase gene of Escherichia coli K12. Primary structure and homology within a family of aminoacyl-TRNA synthetases

The DNA nucleotide sequence of the valS gene encoding valyl-tRNA synthetase of Escherichia coli has been determined. The deduced primary structure of valyl-tRNA synthetase was compared to the primary sequences of the known aminoacyl-tRNA synthetases of yeast and bacteria. Significant homology was detected between valyl-tRNA synthetase of E. coli and other known branched-chain aminoacyl-tRNA synthetases. In pairwise comparisons the highest level of homology was detected between the homologous valyl-tRNA synthetases of yeast and E. coli, with an observed 41% direct identity overall. Comparisons between the valyl- and isoleucyl-tRNA synthetases of E. coli yielded the highest level of homology detected between heterologous enzymes (19.2% direct identity overall). An alignment is presented between the three branched-chain aminoacyl-tRNA synthetases (valyl- and isoleucyl-tRNA synthetases of E. coli and yeast mitochondrial leucyl-tRNA synthetase) illustrating the close relatedness of these enzymes. These results give credence to the supposition that the branched-chain aminoacyl-tRNA synthetases along with methionyl-tRNA synthetase form a family of genes within the aminoacyl-tRNA synthetases that evolved from a common ancestral progenitor gene.     

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.     

Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases

The gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli and adjacent regulatory regions was isolated and sequenced. The structural gene encodes a protein of 471 amino acids whose molecular weight is 53,810. The codon usage is that of genes highly expressed in E. coli. The amino acid sequence deduced from the nucleotide sequence of the gltX gene was confirmed by mass spectrometry of large peptides derived from the glutamyl-tRNA synthetase. The observed peptides confirm 73% of the predicted sequence, including the NH2-terminal and the COOH-terminal segments. Sequence homology between the glutamyl-tRNA synthetase and other aminoacyl-tRNA synthetases of E. coli was found in four segments. Three of them are aligned in the same order in all the synthetases where they are present, but the intersegment spacings are not constant; these ordered segments may come from a progenitor to which other domains were added. Starting from the NH2-end, the first two segments are part of a longer region of homology with the glutaminyl-tRNA synthetase, without need for gaps; its size, about 100 amino acids, is typical of a single folding domain. In the first segment, containing sequences homologous to the HIGH consensus, the homology is consistent with the following evolutionary linkage: gltX----glnS----metS----ileS and tyrS.     

Structure of the yeast isoleucyl-tRNA synthetase gene (ILS1). DNA-sequence, amino-acid sequence of proteolytic peptides of the enzyme and comparison of the structure to those of other known aminoacyl-tRNA synthetases

The ILS1 gene encoding for cytoplasmic isoleucyl-tRNA synthetase from Saccharomyces cerevisiae was subcloned from a 5.4-kb insert of the shuttle vector YEp13 to M13mp8 and M13mp9. Nucleotide sequence analysis of a 4.3-kb BamHI-HpaI fragment revealed a single open reading frame from which we deduced the amino-acid sequence of the enzyme. Independently obtained amino-acid sequence information from ten tryptic peptides of the purified enzyme confirmed the gene-derived structure. The enzyme is comprised of 1073 amino-acids consistent with earlier determinations of its molecular mass. The codon usage of ILS1 is typical of abundant yeast proteins. A significant homology to E. coli isoleucyl- and valyl-tRNA synthetases as well as to yeast valyl-tRNA synthetase was detected. The characteristic amino-acid residues of the aminoacyl-adenylate site and of the potential binding site of the 3'-end of tRNA found in other synthetases are present in the structure.     

Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases from an extreme thermophile Thermus thermophilus HB8: protein structure and Zn2+ binding

Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases have been purified from an extreme thermophile, Thermus thermophilus HB8. Valyl-tRNA and isoleucyl-tRNA synthetases are found to be monomer proteins (Mr 108000 and 129000, respectively), while methionyl-tRNA synthetase is a dimer protein (Mr 150000). These enzymes are very similar with respect to amino acid compositions and alpha-helix contents as estimated by circular dichroism analyses. Furthermore, two Zn2+ are tightly bound to each of these synthetases. These data suggest that valyl-tRNA and isoleucyl-tRNA synthetases consist of two domains, each corresponding to the subunit of methionyl-tRNA synthetase.     

Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway

The Aspergillus nidulans gene (acvA) encoding the first catalytic steps of penicillin biosynthesis that result in the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), has been positively identified by matching a 15-amino acid segment of sequence obtained from an internal CNBr fragment of the purified amino-terminally blocked protein with that predicted from the DNA sequence. acvA is transcribed in the opposite orientation to ipnA (encoding isopenicillin N synthetase), with an intergenic region of 872 nucleotides. The gene has been completely sequenced at the nucleotide level and found to encode a protein of 3,770 amino acids (molecular mass, 422,486 Da). Both fast protein liquid chromatography and native gel estimates of molecular mass are consistent with this predicted molecular weight. The enzyme was identified as a glycoprotein by means of affinity blotting with concanavalin A. No evidence for the presence of introns within the acvA gene has been found. The derived amino acid sequence of ACV synthetase (ACVS) contains three homologous regions of about 585 residues, each of which displays areas of similarity with (i) adenylate-forming enzymes such as parsley 4-coumarate-CoA ligase and firefly luciferase and (ii) several multienzyme peptide synthetases, including bacterial gramicidin S synthetase 1 and tyrocidine synthetase 1. Despite these similarities, conserved cysteine residues found in the latter synthetases and thought to be essential for the thiotemplate mechanism of peptide biosynthesis have not been detected in the ACVS sequence. These observations, together with the occurrence of putative 4'-phosphopantetheine-attachment sites and a putative thioesterase site, are discussed with reference to the reaction sequence leading to production of the ACV tripeptide. We speculate that each of the homologous regions corresponds to a functional domain that recognizes one of the three substrate amino acids.     

Nucleotide sequence of yeast gene CP A1 encoding the small subunit of arginine-pathway carbamoyl-phosphate synthetase. Homology of the deduced amino acid sequence to other glutamine amidotransferases

A yeast DNA fragment carrying the gene CP A1 encoding the small subunit of the arginine pathway carbamoyl-phosphate synthetase has been sequenced. Only one continuous coding sequence on this fragment was long enough to account for the presumed molecular mass of CP A1 protein product. It codes for a polypeptide of 411 amino acids having a relative molecular mass, Mr, of 45 358 and showing extensive homology with the product of carA, the homologous Escherichia coli gene. CP A1 and carA products are glutamine amidotransferases which bind glutamine and transfer its amide group to the large subunits where it is used for the synthesis of carbamoyl-phosphate. A comparison of the amino acid sequences of CP A1 polypeptide with the glutamine amidotransferase domains of anthranilate and p-amino-benzoate synthetases from various sources has revealed the presence in each of these sequences of three highly conserved regions of 8, 11 and 6 amino acids respectively. The 11-residue oligopeptide contains a cysteine which is considered as the active-site residue involved in the binding of glutamine. The distances (number of amino acid residues) which separate these homology regions are accurately conserved in these various enzymes. These observations provide support for the hypothesis that these synthetases have arisen by the combination of a common ancestral glutamine amidotransferase subunit with distinct ammonia-dependent synthetases. Little homology was detected with the amide transfer domain of glutamine phosphoribosyldiphosphate amidotransferase which may be the result of a convergent evolutionary process. The flanking regions of gene CP A1 have been sequenced, 803 base pairs being determined on the 5' side and 382 on the 3' side. Several features of the 5'-upstream region of CP A1 potentially related to the control of its expression have been noticed including the presence of two copies of the consensus sequence d(T-G-A-C-T-C) previously identified in several genes subject to the general control of amino acid biosynthesis.     

Isoleucyl-tRNA synthetase of Methanobacterium thermoautotrophicum Marburg. Cloning of the gene, nucleotide sequence, and localization of a base change conferring resistance to pseudomonic acid

The ileS gene encoding the isoleucyl-tRNA synthetase of the thermophilic archaebacterium Methanobacterium thermoautotrophicum Marburg was isolated and sequenced. ileS was closely flanked by an unknown open reading frame and by purL and thus is arranged differently from the organizations observed in several eubacteria or in Saccharomyces cerevisiae. The deduced amino acid sequence of isoleucyl-tRNA synthetase was compared with primary sequences of isoleucyl-, valyl-, leucyl-, and methionyl-tRNA synthetases from eubacteria and yeast. The archaebacterial enzyme fitted well into this group of enzymes. It contained the two short consensus sequences observed in class I aminoacyl-tRNA synthetases as well as regions of homology with enzymes of the isoleucine family. Comparison between the isoleucyl-tRNA synthetases of M. thermoautotrophicum yielded 36% amino acid identity with the yeast enzyme and 32% identity with the corresponding enzyme from Escherichia coli. The ileS gene of the pseudomonic acid-resistant M. thermoautotrophicum mutant MBT10 was also sequenced. The mutant enzyme had undergone a glycine to aspartic acid transition at position 590, in a conserved region comprising the KMSKS consensus sequence. The inhibition constants of pseudomonic acid, KiIle and KiATP, for the mutant enzyme were 10-fold higher than those determined for the wild-type enzyme. Both the mutant and the wild-type ileS gene were expressed in E. coli, and their products displayed the expected difference in sensitivity toward pseudomonic acid.     

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.     

Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state.     

Nuclear overhauser effect studies of the conformations of Mg(alpha, beta-methylene)ATP bound to E. coli isoleucyl-tRNA synthetase

Internuclear distances obtained from transferred nuclear Overhauser effects were used in combination with distance geometry calculations to define the E. coli isoleucyl-tRNA synthetase bound conformation of Mg(alpha, beta-methylene)ATP both in the absence and in the presence of the cognate and noncognate amino acids L-isoleucine and L-valine, respectively. A single nucleotide structure having an anti adenine-ribose glycosidic torsional angle of -114 degrees was found to satisfy the experimental distance constraints. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrate that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or to the nature of the amino acid bound at the aminoacyladenylate site. In addition, the acceptable range of Mg(alpha, beta-methylene)ATP conformations bound to the E. coli isoleucyl-tRNA synthetase was found to be nearly identical to that previously determined for the E. coli methionyl-tRNA synthetase (Williams and Rosevear (1991) J. Biol. Chem. 266, 2089-2098). Thus, the predicted structural homology between the isoleucyl- and methionyl-tRNA synthetases, both members of the same class of synthetases on the basis of common consensus sequences, is further supported by consensus enzyme-bound nucleotide conformations.     

Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence

Glutamine synthetase is encoded by the glnA gene of Escherichia coli and catalyzes the formation of glutamine from ATP, glutamate, and ammonia. A 1922-base pair fragment from a cDNA containing the glnA structural gene for E. coli glutamine synthetase has been sequenced. An open reading frame of 1404 base pairs encodes a protein of 468 amino acid residues with a calculated molecular weight of 51,814. With few exceptions, the amino acid sequence deduced from the DNA sequence agreed very well with the amino acid sequences of several peptides reported previously. The secondary structure predicted for the E. coli enzyme has approximately 36% of the residues in alpha-helices which is in agreement with calculations of approximately 39% based on optical rotatory dispersion data. Comparison of the amino acid sequences of glutamine synthetase from E. coli (468 amino acids) and Anabaena (473 amino acids) (Turner, N. E., Robinson, S. T., and Haselkorn, R. (1983) Nature 306, 337-342) indicates that 260 amino acids are identical and 80 are of the same type (polar or nonpolar) when aligned for maximum homology. Several homologous regions of these two enzymes exist, including the sites of adenylylation and oxidative modification, but the regulation of each enzyme is different.     

Expression and characterization of the human mitochondrial leucyl-tRNA synthetase

A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeuRS) has been identified from the EST databases. Analysis of the protein encoded by this cDNA indicates that the protein is 903 amino acids in length and contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl-tRNA synthetases and regions of high homology to other mitochondrial and bacterial LeuRS proteins. The mature form of this protein has been cloned and expressed in Escherichia coli. Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa. The human mtLeuRS is capable of aminoacylating E. coli tRNA(Leu). Its activity is inhibited at high levels of either monovalent or divalent cations. K(M) and k(cat) values for ATP:PP(i) exchange and for the aminoacylation reaction have been determined.     

The macrophage-induced gene (mig) of Mycobacterium avium encodes a medium-chain acyl-coenzyme A synthetase.

The macrophage-induced gene (mig) of Mycobacterium avium has been associated with virulence, but the functions of the gene product were still unknown. Here we have characterized the Mig protein by biochemical methods. A plasmid with a histidine-tagged fusion protein was constructed for expression in Escherichia coli. Mig was detected as a 60 kDa protein after expression and purification of the recombinant gene product. The sequence of the fusion gene and of the parent gene in M. avium were reexamined. This confirmed that the mig gene encodes a 550 amino acid protein (58 kDa) instead of a 295 amino acid protein (30 kDa) as predicted before. The 550 amino acid Mig exhibits a high degree of homology to bacterial acyl-CoA synthetases. Two artificial 30 kDa derivatives of Mig were expressed and purified as histidine-tagged fusion proteins in E. coli. These proteins and the 58.6 kDa histidine-tagged Mig protein were analysed for activity with an acyl-CoA synthetase assay. Among the three investigated proteins, only the 58.6 kDa Mig exhibited detectable activity as an acyl-CoA synthetase (EC 6.2.1.3) with saturated medium-chain fatty acids, unsaturated long-chain fatty acid and some aromatic carbon acids as substrates. Enzymatic activity could be inhibited by 2-hydroxydodecanoic acid, a typical inhibitor of medium-chain acyl-CoA synthetases. We postulate a novel medium-chain acyl-CoA synthetase motif. We have investigated the biochemical properties of Mig and suggest that this enzyme is involved in the metabolism of fatty acid during mycobacterial survival in macrophages.     

Identification of a long-chain polyunsaturated fatty acid acyl-coenzyme A synthetase from the diatom Thalassiosira pseudonana

The draft genome of the diatom Thalassiosira pseudonana was searched for DNA sequences showing homology with long-chain acyl-coenzyme A synthetases (LACSs), since the corresponding enzyme may play a key role in the accumulation of health-beneficial polyunsaturated fatty acids (PUFAs) in triacylglycerol. Among the candidate genes identified, an open reading frame named TplacsA was found to be full length and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpLACSA, exhibited typical features of acyl-coenzyme A (acyl-CoA) synthetases involved in the activation of long-chain fatty acids. Feeding experiments carried out in yeast (Saccharomyces cerevisiae) transformed with the algal gene showed that TpLACSA was able to activate a number of PUFAs, including eicosapentaenoic acid and docosahexaenoic acid (DHA). Determination of acyl-CoA synthetase activities by direct measurement of acyl-CoAs produced in the presence of different PUFA substrates showed that TpLACSA was most active toward DHA. Heterologous expression also revealed that TplacsA transformants were able to incorporate more DHA in triacylglycerols than the control yeast.     

Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps

Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps.