Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [(32)P]pyrophosphate-ATP exchange technique. 3. beta-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and gamma-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for beta-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. alpha-Cyclopropylglycine and alpha-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase.
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PMID:Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species. 549 5

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

In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.
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PMID:Towards a phosphoproteome map of Corynebacterium glutamicum. 1292 88

Aminoacyl-tRNA synthetases (aaRSs) strictly discriminate their cognate amino acids. Some aaRSs accomplish this via proofreading and editing mechanisms. Mursinna and coworkers recently reported that substituting a highly conserved threonine (T252) with an alanine within the editing domain of Escherichia coli leucyl-tRNA synthetase (LeuRS) caused LeuRS to cleave its cognate aminoacylated leucine from tRNA(Leu) (Mursinna et al., Biochemistry 2001;40:5376-5381). To achieve atomic level insight into the role of T252 in LeuRS and the editing reaction of aaRSs, a series of molecular modeling studies including homology modeling and automated docking simulations were carried out. A 3D structure of E. coli LeuRS was constructed via homology modeling using the X-ray structure of Thermus thermophilus LeuRS as a template because the E. coli LeuRS structure is not available from X-ray or NMR studies. However, both the X-ray T. thermophilus and homology-modeled E. coli structures were used in our studies. Amino acid binding sites in the proposed editing domain, which is also called the connective polypeptide 1 (CP1) domain, were investigated by automated docking studies. The root mean square deviation (RMSD) for backbone atoms between the X-ray and homology-modeled structures was 1.18 A overall and 0.60 A for the editing (CP1) domain. Automated docking studies of a leucine ligand into the editing domain were performed for both structures: homology structure of E. coli LeuRS and X-ray structure of T. thermophilus LeuRS for comparison. The results of the docking studies suggested that there are two possible amino acid binding sites in the CP1 domain for both proteins. The first site lies near a threonine-rich region that includes the highly conserved T252 residue, which is important for amino acid discrimination. The second site is located in a flexible loop region surrounded by residues E292, A293, M295, A296, and M298. The important T252 residue is at the bottom of the first binding pocket.
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PMID:Molecular modeling study of the editing active site of Escherichia coli leucyl-tRNA synthetase: two amino acid binding sites in the editing domain. 1499 65

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

Some aaRSs (aminoacyl-tRNA synthetases) develop editing mechanisms to correct mis-charged tRNA. The CP1 (connective peptide 1) domain of LeuRS (leucyl-tRNA synthetase) contains the editing active site, which is the proven target for the broad-spectrum drug AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole). The ESI (eukarya-specific insertion 1) in the CP1 domain of GlLeuRS (Giardia lamblia LeuRS) has been identified. Similar substitution with the ESI from HsLeuRS (Homo sapiens LeuRS) impeded the leucine activation, aminoacylation and post-transfer editing of the enzyme, but had no effect on the editing specificity toward non-specific amino acids. Thr341 in GlLeuRS served as a specificity discriminator, as found in other LeuRS systems, although its substitution with an alanine residue did not destroy Leu-tRNALeu synthesis in vitro and in vivo. The Arg338 was crucial for tRNALeu charging and the Asp440 was crucial for leucine activation and aminoacylation. The post-transfer editing required the CTD (C-terminal domain), Arg338 and Asp440 of GlLeuRS. Interestingly, GlLeuRS was completely resistant to the AN2690, which is an inhibitor of various LeuRSs. The universally conserved aspartate residue in the LeuRS CP1 domains was responsible for the resistance of GlLeuRS and another recently reported AN2690-resistant AaLeuRS (Aquifex aeolicus LeuRS). Our results indicate the functional divergence of some absolutely conserved sites, improve the understanding of the editing function of eukaryotic/archaeal LeuRSs and shed light on the development of a GlLeuRS-specific inhibitor for the treatment of giardiasis.
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PMID:Post-transfer editing by a eukaryotic leucyl-tRNA synthetase resistant to the broad-spectrum drug AN2690. 2055 93

The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.
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PMID:Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase. 2352 58