scholarly journals Oxidation alters the architecture of the phenylalanyl-tRNA synthetase editing domain to confer hyperaccuracy

2021 ◽  
Author(s):  
Pooja Srinivas ◽  
Rebecca E Steiner ◽  
Ian J Pavelich ◽  
Ricardo Guerrero-Ferreira ◽  
Puneet Juneja ◽  
...  
Keyword(s):  
Salmonella Enterica Serovar Typhimurium ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Human Infection ◽  
Hydroxyl Group ◽  
High Fidelity ◽  
Trna Synthetases ◽  
Cryo Electron Microscopy ◽  
Serovar Typhimurium ◽  
Editing Domain

Abstract High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the β-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.

scholarly journals Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination

2006 ◽  
Vol 394 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Yunqing Liu ◽  
Jing Liao ◽  
Bin Zhu ◽  
En-Duo Wang ◽  
Jianping Ding
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Vital Role ◽  
Common Mechanism ◽  
Trna Synthetases ◽  
Editing Domain

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.

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.

scholarly journals Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

Molecules ◽  
2020 ◽  
Vol 25 (19) ◽  
pp. 4418
Author(s):  
Hsueh-Wei Tseng ◽  
Tobias Baumann ◽  
Huan Sun ◽  
Yane-Shih Wang ◽  
Zoya Ignatova ◽  
...  
Keyword(s):  
Amino Acids ◽  
Aromatic Amino Acids ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Rational Approach ◽  
Translation Efficiency ◽  
Amino Acid Residues ◽  
Trna Synthetases ◽  
Methanosarcina Mazei ◽  
Genetic Code Expansion

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation

Human cytoplasmic ProX edits mischarged tRNAPro with amino acid but not tRNA specificity

2013 ◽  
Vol 450 (1) ◽  
pp. 243-252 ◽  
Author(s):  
Liang-Liang Ruan ◽  
Xiao-Long Zhou ◽  
Min Tan ◽  
En-Duo Wang
Keyword(s):  
Amino Acid ◽  
Protein Biosynthesis ◽  
Trna Synthetase ◽  
Main Body ◽  
Amino Acid Residues ◽  
Homologous Proteins ◽  
Gene Encoding ◽  
Trna Synthetases ◽  
Editing Domain ◽  
First Time

aaRSs (aminoacyl-tRNA synthetases) are responsible for ensuring the fidelity of the genetic code translation by accurately linking a particular amino acid to its cognate tRNA isoacceptor. To ensure accuracy of protein biosynthesis, some aaRSs have evolved an editing process to remove mischarged tRNA. The hydrolysis of the mischarged tRNA usually occurs in an editing domain, which is inserted into or appended to the main body of the aaRS. In addition, autonomous, editing domain-homologous proteins can also trans-edit mischarged tRNA in concert or in compensating for the editing function of its corresponding aaRS. The freestanding ProX is a homologue of the editing domain of bacterial ProRS (prolyl-tRNA synthetase). In the present study, we cloned for the first time a gene encoding HsProX (human cytoplasmic ProX) and purified the expressed recombinant protein. The catalytic specificity of HsProX for non-cognate amino acids and identity elements on tRNAPro for editing were also investigated. We found that HsProX could deacylate mischarged Ala-tRNAPro, but not Cys-HstRNAUGGPro, and specifically targeted the alanine moiety of Ala-tRNAPro. The importance of the CCA76 end of the tRNA for deacylation activity and key amino acid residues in HsProX for its editing function were also identified.

scholarly journals Aspects of Nucleic Acid Structure and Function

2020 ◽  
pp. 31-40
Author(s):  
Sosale Chandrasekhar
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Genetic Code ◽  
Rna World ◽  
Hydrolytic Stability ◽  
Trna Synthetase ◽  
Hydroxyl Group ◽  
Effect Of Temperature ◽  
Structural Basis ◽  
Trna Synthetases

Despite decades of intense study, certain physico-chemical aspects of the nucleic acids–the genetic repository of life–remain enigmatic. Thus, solutions of DNA are apparently constituted of varying amounts of double and single-stranded forms, with melting studies being inconclusive about the effect of temperature on the composition. Consequently, this casts doubt on current estimates of the thermodynamic stability of the base pairs. However, the overwhelming stability of the Watson-Crick model is adumbrated by a kinetic analysis of the action of DNA polymerase, microscopic reversibility indicating that polymerase action is kinetically controlled, whereas proof reading-excision is thermodynamically controlled. The structural basis for the differing roles played by DNA and RNA in the sustenance and propagation of life also remains to be clarified. It appears that formation of the RNA double helix is sterically inhibited by the 2’ ribose hydroxyl group, which is also relatively inaccessible to external base, thus enhancing hydrolytic stability. The in vivo conformation of RNA is likely determined by the requirements of its appointed biological role, the large size of tRNAs being particularly significant in possibly leading to enhanced specificity in its interaction with the corresponding tRNA synthetase. The proclivity of RNA generally to remain single stranded may indeed be the reason for the existence of viruses as stable RNA-protein complexes. The preeminent role of nucleic acids as “genetic guardians” is, however, blurred by the fact that the translation of the genetic code is contingent on the action of the various tRNA synthetases. This implies that the genetic code is manifested via codon-anticodon specificity only by the involvement of protein molecules that are unique to each codon-anticodon pair. This has intriguing implications for the origin and evolution of the genetic code, possibly indicating that the tRNA synthetases are a relic of prebiotic protein-nucleic acid hybrids (thus also raising doubts about the RNA world 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 Outbreak of unusual Salmonella enterica serovar Typhimurium monophasic variant 1,4 [5],12:i:-, Italy, June 2013 to September 2014

2016 ◽  
Vol 21 (15) ◽  
Author(s):  
Francesca Cito ◽  
Francesca Baldinelli ◽  
Paolo Calistri ◽  
Elisabetta Di Giannatale ◽  
Gaia Scavia ◽  
...  
Keyword(s):  
Surface Water ◽  
Salmonella Enterica ◽  
Environmental Samples ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Central Italy ◽  
Human Infection ◽  
Antigenic Structure ◽  
The Third ◽  
Abruzzo Region ◽  
Serovar Typhimurium

Monophasic variant of Salmonella enterica subspecies enterica serovar Typhimurium (monophasic S. Typhimurium), with antigenic structure 1,4,[5],12:i:-, appears to be of increasing importance in Europe. In Italy, monophasic S. Typhimurium represented the third most frequent Salmonella serovar isolated from human cases between 2004 and 2008. From June 2013 to October 2014, a total of 206 human cases of salmonellosis were identified in Abruzzo region (Central Italy). Obtained clinical isolates characterised showed S. Typhimurium 1,4,[5],12:i:- with sole resistance to nalidixic acid, which had never been observed in Italy in monophasic S. Typhimurium, neither in humans nor in animals or foods. Epidemiological, microbiological and environmental investigations were conducted to try to identify the outbreak source. Cases were interviewed using a standardised questionnaire and microbiological tests were performed on human as well as environmental samples, including samples from fruit and vegetables, pigs, and surface water. Investigation results did not identify the final vehicle of human infection, although a link between the human cases and the contamination of irrigation water channels was suggested.

scholarly journals CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS

2021 ◽  
Vol 118 (13) ◽  
pp. e2012898118
Author(s):  
Litao Sun ◽  
Na Wei ◽  
Bernhard Kuhle ◽  
David Blocquel ◽  
Scott Novick ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Hydrodynamic Diameter ◽  
Mutant Proteins ◽  
X Ray ◽  
Trna Synthetases ◽  
X Ray Crystallography ◽  
Charcot Marie Tooth Disease ◽  
Neuropilin 1 ◽  
Editing Domain

Through dominant mutations, aminoacyl-tRNA synthetases constitute the largest protein family linked to Charcot-Marie-Tooth disease (CMT). An example is CMT subtype 2N (CMT2N), caused by individual mutations spread out in AlaRS, including three in the aminoacylation domain, thereby suggesting a role for a tRNA-charging defect. However, here we found that two are aminoacylation defective but that the most widely distributed R329H is normal as a purified protein in vitro and in unfractionated patient cell samples. Remarkably, in contrast to wild-type (WT) AlaRS, all three mutant proteins gained the ability to interact with neuropilin 1 (Nrp1), the receptor previously linked to CMT pathogenesis in GlyRS. The aberrant AlaRS-Nrp1 interaction is further confirmed in patient samples carrying the R329H mutation. However, CMT2N mutations outside the aminoacylation domain do not induce the Nrp1 interaction. Detailed biochemical and biophysical investigations, including X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter determinations, and protease digestions reveal a mutation-induced structural loosening of the aminoacylation domain that correlates with the Nrp1 interaction. The b1b2 domains of Nrp1 are responsible for the interaction with R329H AlaRS. The results suggest Nrp1 is more broadly associated with CMT-associated members of the tRNA synthetase family. Moreover, we revealed a distinct structural loosening effect induced by a mutation in the editing domain and a lack of conformational impact with C-Ala domain mutations, indicating mutations in the same protein may cause neuropathy through different mechanisms. Our results show that, as with other CMT-associated tRNA synthetases, aminoacylation per se is not relevant to the pathology.

scholarly journals Duplication of leucyl-tRNA synthetase in an archaeal extremophile may play a role in adaptation to variable environmental conditions

2020 ◽  
Vol 295 (14) ◽  
pp. 4563-4576 ◽  
Author(s):  
Christopher S. Weitzel ◽  
Li Li ◽  
Changyi Zhang ◽  
Kristen K. Eilts ◽  
Nicholas M. Bretz ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Environmental Conditions ◽  
Active Sites ◽  
Hot Springs ◽  
Protein Translation ◽  
Selective Advantage ◽  
Trna Synthetase ◽  
Bioinformatics Analyses ◽  
Trna Synthetases ◽  
Editing Domain

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that play a fundamental role in protein synthesis. They catalyze the esterification of specific amino acids to the 3′-end of their cognate tRNAs and therefore play a pivotal role in protein synthesis. Although previous studies suggest that aaRS-dependent errors in protein synthesis can be beneficial to some microbial species, evidence that reduced aaRS fidelity can be adaptive is limited. Using bioinformatics analyses, we identified two distinct leucyl-tRNA synthetase (LeuRS) genes within all genomes of the archaeal family Sulfolobaceae. Remarkably, one copy, designated LeuRS-I, had key amino acid substitutions within its editing domain that would be expected to disrupt hydrolytic editing of mischarged tRNALeu and to result in variation within the proteome of these extremophiles. We found that another copy, LeuRS-F, contains canonical active sites for aminoacylation and editing. Biochemical and genetic analyses of the paralogs within Sulfolobus islandicus supported the hypothesis that LeuRS-F, but not LeuRS-I, functions as an essential tRNA synthetase that accurately charges leucine to tRNALeu for protein translation. Although LeuRS-I was not essential, its expression clearly supported optimal S. islandicus growth. We conclude that LeuRS-I may have evolved to confer a selective advantage under the extreme and fluctuating environmental conditions characteristic of the volcanic hot springs in which these archaeal extremophiles reside.

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