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Query: EC:6.1.1.4 (leucyl-tRNA synthetase)
 297 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CP1) inserted into its nucleotide binding fold, or active site. In this study, purified leucyl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptides could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP approximately PP(i) exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region around the bond of E292 and A293, four pairs of peptides resulting from different cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cleaved LeuRS could not catalyze aminoacylation, fluorescence titration revealed that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be responsible for maintaining the proper conformation of LeuRS required for the tRNA charging activity.
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PMID:The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity. 1052 76

The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed. This "double-sieve" mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function.
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PMID:CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function. 1082 91

Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accurately selecting and activating cognate amino acids for aminoacylation of the correct tRNA. Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection. These two sieves rely on different strategies for amino acid recognition to significantly enhance the accuracy of aminoacylation. We have performed alanine scanning mutagenesis in a conserved threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's CP1 domain that is hypothesized to contain a putative editing active site. Characterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid from being hydrolyzed after aminoacylation of the tRNA. Mutation of this threonine to an alanine eliminates discrimination of the cognate amino acid in the editing active site. This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site.
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PMID:A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu. 1133 Oct

Yeast mitochondrial leucyl-tRNA synthetase (LeuRS) binds to the bI4 intron and collaborates with the bI4 maturase to aid excision of the group I intron. Deletion analysis isolated the inserted LeuRS CP1 domain as a critical factor in the protein's splicing activity. Protein fragments comprised of just the LeuRS CP1 region rescued complementation of a yeast strain that expressed a splicing-defective LeuRS. Three-hybrid analysis determined that these CP1-containing LeuRS fragments, ranging from 214 to 375 amino acids, bound to the bI4 intron. In each case, interactions with only the LeuRS protein fragment specifically stimulated bI4 intron splicing activity. Substitution of a homologous CP1 domain from isoleucyl-tRNA synthetase or mutation within the LeuRS CP1 region of the smallest protein fragment abolished RNA binding and splicing activity. The CP1 domain is best known for its amino acid editing activity. However, these results suggest that elements within the LeuRS CP1 domain also play a novel role, independent of the full-length tRNA synthetase, in binding the bI4 group I intron and facilitating its self-splicing activity.
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PMID:An inserted region of leucyl-tRNA synthetase plays a critical role in group I intron splicing. 1248 8

Escherichia coli leucyl-tRNA synthetase (LeuRS) belongs to class I aminoacyl-tRNA synthetases. It consists of 860 amino acid residues and catalyzes the leucylation of tRNA(Leu). An insertion of its 253-368 peptide fragment between 368 to 369 in CP1 domain of this enzyme was shown to maintain the activity of the enzyme, and the insertion mutant was named as LeuRS-C. Because the insertion mutant of LeuRS was sensitive to operation of the purification, a plasmid containing the gene encoding LeuRS with His(6)-tag at its N-terminus was constructed to facilitate the purification of His(6)-LeuRS-C through one-step affinity chromatography on Ni(2+)-NTA column. The purified His(6)-LeuRS-C had full activity as the native LeuRS with His-tag at the N-terminus (His(6)-LeuRS), although the mutant enzyme had an insertion of 116 amino acid residues. The kinetic parameters of His(6)-LeuRS-C were determined. The secondary structure estimated by CD spectrum and thermal stability of the insertion mutant was compared with those of His(6)-LeuRS, respectively.
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PMID:[An insertion mutant of LeuRS with 116 amino acid residues has full activity]. 1262 46

Escherichia coli leucyl-tRNA synthetase (LeuRS) has a large connecting polypeptide (CP1) inserted into its active site. It was demonstrated that the peptide bond between E292-A293 was crucial for the aminoacylation activity of E. coli LeuRS. To investigate the effect of E292 on the function of Escherichia coli LeuRS, E292 was mutated to K, F, S, D, Q and A. These mutations at 292 did not change the specific activity of the amino acid activation reaction. Though the conformational change of these mutants was not detected in CD, their aminoacylation activities were impaired to varying extents. The mutation of E to K decreased the aminoacylation activity to the largest extent. Analysis of the Km values of these mutants for the three substrates showed that the E292 was not involved in the binding of leucine and that all mutants had stronger binding with ATP.
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PMID:E292 is important for the aminoacylation activity of Escherichia coli leucyl-tRNA synthetase. 1273

Isoleucyl-tRNA synthetase (IleRS) links tRNA(Ile) with not only its cognate isoleucine but also the nearly cognate valine. The CP1 domain of IleRS deacylates, or edits, the mischarged Val-tRNA(Ile). We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-A resolutions, respectively. In the complex structure, the Asp(328) residue, which was shown to be critical for the editing reaction against Val-tRNA(Ile) by a previous mutational analysis, recognizes the valine NH(3)(+) group. The valine side chain binding pocket is only large enough to accommodate valine, and the placement of an isoleucine model in this location revealed that the additional methylene group of isoleucine would clash with His(319). The H319A mutant of Escherichia coli IleRS reportedly deacylates the cognate Ile-tRNA(Ile) in addition to Val-tRNA(Ile), indicating that the valine-binding mode found in this study represents that in the Val-tRNA(Ile) editing reaction. Analyses of the Val-tRNA(Ile) editing activities of T. thermophilus IleRS mutants revealed the importance of Thr(228), Thr(229), Thr(230), and Asp(328), which are coordinated with water molecules in the present structure. The structural model for the Val-adenosine moiety of Val-tRNA(Ile) bound in the IleRS editing site revealed some interesting differences in the substrate binding and recognizing mechanisms between IleRS and T. thermophilus leucyl-tRNA synthetase. For example, the carbonyl oxygens of the amino acids are located opposite to each other, relative to the adenosine ribose ring, and are differently recognized.
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PMID:Crystal structures of the CP1 domain from Thermus thermophilus isoleucyl-tRNA synthetase and its complex with L-valine. 1467 40

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.
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PMID:Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism. 1630 Mar 91

The editing or hydrolytic CP1 domain of leucyl-tRNA synthetase (LeuRS) hydrolyses several misactivated amino acids. The CP1 domain of Aquifex aeolicus LeuRS was expressed, purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.8, b = 98.4, c = 116.7 A. Crystals diffract to beyond 1.8 A resolution and contain two monomers in the asymmetric unit. Two CP1 mutants in which a conserved threonine residue essential for the fidelity of the hydrolytic pathway is mutated to alanine or glutamic acid have also been expressed and crystallized. Crystals of the two CP1 mutants are isomorphs of the wild type and diffract to beyond 1.9 A resolution. All structures were solved by molecular-replacement techniques.
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PMID:Crystallization and preliminary X-ray crystallographic study of the wild type and two mutants of the CP1 hydrolytic domain from Aquifex aeolicus leucyl-tRNA synthetase. 1651 Nov 90

Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase.
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PMID:A viable amino acid editing activity in the leucyl-tRNA synthetase CP1-splicing domain is not required in the yeast mitochondria. 1695 79


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