A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

2012 ◽  
Vol 443 (2) ◽  
pp. 477-484 ◽  
Author(s):  
Min Tan ◽  
Wei Yan ◽  
Ru-Juan Liu ◽  
Meng Wang ◽  
Xin Chen ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Trna Synthetase ◽  
Modular Construction ◽  
Aquifex Aeolicus ◽  
Trna Synthetases ◽  
Naturally Occurring ◽  
Aminoacyl Trna Synthetases ◽  
Editing Domain ◽  
Editing Activity ◽  
Shed Light

aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain fromAeropyrum pernix

2012 ◽  
Vol 68 (11) ◽  
pp. 1390-1393 ◽  
Author(s):  
Sadeem Ahmad ◽  
Antony S. K. Sravankumar ◽  
Shobha P. Kruparani ◽  
Rajan Sankaranarayanan
Keyword(s):  
Trna Synthetase ◽  
Asymmetric Unit ◽  
Crystallographic Investigation ◽  
X Ray ◽  
Trna Synthetases ◽  
Aeropyrum Pernix ◽  
Substrate Analogues ◽  
Aminoacyl Trna Synthetases ◽  
Editing Domain ◽  
Evolutionary Aspects

The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeonPyrococcus abysiiin complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeonAeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase,i.e.ThrRS1 and ThrRS2, encoded by two different genes,thrS1andthrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 fromA. pernixis reported. The crystals belong to either theP41212 orP43212 space group and consist of one monomer per asymmetric unit.

Peripheral insertion modulates the editing activity of the isolated CP1 domain of leucyl-tRNA synthetase

2011 ◽  
Vol 440 (2) ◽  
pp. 217-227 ◽  
Author(s):  
Ru-Juan Liu ◽  
Min Tan ◽  
Dao-Hai Du ◽  
Bei-Si Xu ◽  
Gilbert Eriani ◽  
...  
Keyword(s):  
Active Site ◽  
Trna Synthetase ◽  
Site Directed Mutagenesis ◽  
Aquifex Aeolicus ◽  
Trna Synthetases ◽  
Catalytic Function ◽  
Editing Activity ◽  
Domain Docking ◽  
Dynamics Simulations

A large insertion domain called CP1 (connective peptide 1) present in class Ia aminoacyl-tRNA synthetases is responsible for post-transfer editing. LeuRS (leucyl-tRNA synthetase) from Aquifex aeolicus and Giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the CP1 that are crucial for editing activity. Crystal structures of AaLeuRS-CP1 [2.4 Å (1 Å=0.1 nm)], GlLeuRS-CP1 (2.6 Å) and the insertion deletion mutant AaLeuRS-CP1Δ20 (2.5 Å) were solved to understand the role of these insertions in editing. Both insertions are folded as peripheral motifs located on the opposite side of the proteins from the active-site entrance in the CP1 domain. Docking modelling and site-directed mutagenesis showed that the insertions do not interact with the substrates. Results of molecular dynamics simulations show that the intact CP1 is more dynamic than its mutant devoid of the insertion motif. Taken together, the data show that a peripheral insertion without a substrate-binding site or major structural role in the active site may modulate catalytic function of a protein, probably from protein dynamics regulation in two respective LeuRS CP1s. Further results from proline and glycine mutational analyses intended to reduce or increase protein flexibility are consistent with this hypothesis.

scholarly journals Stereospecificity control in aminoacyl-tRNA-synthetases: new evidence of d-amino acids activation and editing

2019 ◽  
Vol 47 (18) ◽  
pp. 9777-9788
Author(s):  
Mariia Yu Rybak ◽  
Alexey V Rayevsky ◽  
Olga I Gudzera ◽  
Michael A Tukalo
Keyword(s):  
Amino Acids ◽  
Thermus Thermophilus ◽  
Protective Role ◽  
Trna Synthetase ◽  
Evolutionary Strategies ◽  
Computational Docking ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Editing Domain ◽  
Dynamics Simulations

Abstract The homochirality of amino acids is vital for the functioning of the translation apparatus. l-Amino acids predominate in proteins and d-amino acids usually represent diverse regulatory functional physiological roles in both pro- and eukaryotes. Aminoacyl-tRNA-synthetases (aaRSs) ensure activation of proteinogenic or nonproteinogenic amino acids and attach them to cognate or noncognate tRNAs. Although many editing mechanisms by aaRSs have been described, data about the protective role of aaRSs in d-amino acids incorporation remained unknown. Tyrosyl- and alanyl-tRNA-synthetases were represented as distinct members of this enzyme family. To study the potential to bind and edit noncognate substrates, Thermus thermophilus alanyl-tRNA-synthetase (AlaRS) and tyrosyl-tRNA-synthetase were investigated in the context of d-amino acids recognition. Here, we showed that d-alanine was effectively activated by AlaRS and d-Ala-tRNAAla, formed during the erroneous aminoacylation, was edited by AlaRS. On the other hand, it turned out that d-aminoacyl-tRNA-deacylase (DTD), which usually hydrolyzes d-aminoacyl-tRNAs, was inactive against d-Ala-tRNAAla. To support the finding about DTD, computational docking and molecular dynamics simulations were run. Overall, our work illustrates the novel function of the AlaRS editing domain in stereospecificity control during translation together with trans-editing factor DTD. Thus, we propose different evolutionary strategies for the maintenance of chiral selectivity during translation.

scholarly journals Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites

2011 ◽  
Vol 108 (23) ◽  
pp. 9378-9383 ◽  
Author(s):  
L. Li ◽  
M. T. Boniecki ◽  
J. D. Jaffe ◽  
B. S. Imai ◽  
P. M. Yau ◽  
...  
Keyword(s):  
Trna Synthetases ◽  
Naturally Occurring ◽  
Aminoacyl Trna Synthetases ◽  
Editing Domain

scholarly journals Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bingyi Chen ◽  
Siting Luo ◽  
Songxuan Zhang ◽  
Yingchen Ju ◽  
Qiong Gu ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Domain ◽  
Trna Synthetase ◽  
Rational Drug Design ◽  
Class I ◽  
Binding Assays ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Rational Drug ◽  
Trna Binding

AbstractThe polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with l-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Sanya Chadha ◽  
N. Arjunreddy Mallampudi ◽  
Debendra K. Mohapatra ◽  
Rentala Madhubala
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Essential Gene ◽  
Protozoan Parasite ◽  
Protein Translation ◽  
Trna Synthetase ◽  
Parasite Growth ◽  
Coding Sequences ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC50], 4.19 µM) and intracellular amastigotes (IC50, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LdLysRS-1 as a potential drug target.

scholarly journals Influence of Antisynthetase Antibodies Specificities on Antisynthetase Syndrome Clinical Spectrum Time Course

2019 ◽  
Vol 8 (11) ◽  
pp. 2013 ◽  
Author(s):  
Cavagna ◽  
Trallero-Araguás ◽  
Meloni ◽  
Cavazzana ◽  
Rojas-Serrano ◽  
...  
Keyword(s):  
Time Course ◽  
Clinical Presentation ◽  
Reference Group ◽  
Trna Synthetase ◽  
Antisynthetase Syndrome ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Antisynthetase Antibodies ◽  
Clinical Triad

Antisynthetase syndrome (ASSD) is a rare clinical condition that is characterized by the occurrence of a classic clinical triad, encompassing myositis, arthritis, and interstitial lung disease (ILD), along with specific autoantibodies that are addressed to different aminoacyl tRNA synthetases (ARS). Until now, it has been unknown whether the presence of a different ARS might affect the clinical presentation, evolution, and outcome of ASSD. In this study, we retrospectively recorded the time of onset, characteristics, clustering of triad findings, and survival of 828 ASSD patients (593 anti-Jo1, 95 anti-PL7, 84 anti-PL12, 38 anti-EJ, and 18 anti-OJ), referring to AENEAS (American and European NEtwork of Antisynthetase Syndrome) collaborative group’s cohort. Comparisons were performed first between all ARS cases and then, in the case of significance, while using anti-Jo1 positive patients as the reference group. The characteristics of triad findings were similar and the onset mainly began with a single triad finding in all groups despite some differences in overall prevalence. The “ex-novo” occurrence of triad findings was only reduced in the anti-PL12-positive cohort, however, it occurred in a clinically relevant percentage of patients (30%). Moreover, survival was not influenced by the underlying anti-aminoacyl tRNA synthetase antibodies’ positivity, which confirmed that antisynthetase syndrome is a heterogeneous condition and that antibody specificity only partially influences the clinical presentation and evolution of this condition.

Aminoacyl-tRNA synthetases of Halobacterium cutirubrum: preliminary fractionation studies

1970 ◽  
Vol 48 (8) ◽  
pp. 944-946 ◽  
Author(s):  
E. Griffiths
Keyword(s):  
Ionic Strength ◽  
Gel Filtration ◽  
Halophilic Bacterium ◽  
Trna Synthetase ◽  
Partial Purification ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Fractionation Procedure ◽  
The Stability ◽  
Low Ionic Strength

The stability, in solutions of low ionic strength, of aminoacyl-tRNA synthetases from the extremely halophilic bacterium Halobacterium cutirubrum was studied as a preliminary to their fractionation. The enzymes differed considerably in their sensitivity to such solutions. Conditions were found where reactivation from the salt-free and inactive state could be achieved. Removal of both K+ and Mg2+ together generally resulted in better stability than the removal of K+ alone. A low temperature (4°) was also important for stability in buffers of low ionic strength. In some cases the L-amino acid substrates afforded protection against inactivation in the salt-free state. Gel filtration in low ionic strength medium was found to work well as a fractionation procedure; a partial purification of phenylalanyl-tRNA synthetase was effected in this way. The use of other conventional protein fractionation procedures is now possible.

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