Deciphering the interaction of benzoxaborole inhibitor AN2690 with connective polypeptide 1 (CP1) editing domain of Leishmania donovani leucyl-tRNA synthetase

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.

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

mSphere ◽

10.1128/mspheredirect.00340-17 ◽

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.

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Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0603182103 ◽

2006 ◽

Vol 103 (40) ◽

pp. 14744-14749 ◽

Cited By ~ 26

Author(s):

H. M. Sasaki ◽

S.-i. Sekine ◽

T. Sengoku ◽

R. Fukunaga ◽

M. Hattori ◽

...

Keyword(s):

Amino Acid ◽

Trna Synthetase ◽

Amino Acid Editing ◽

Editing Domain

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Strictly Conserved Lysine of Prolyl-tRNA Synthetase Editing Domain Facilitates Binding and Positioning of Misacylated tRNAPro

Biochemistry ◽

10.1021/bi401279r ◽

2014 ◽

Vol 53 (6) ◽

pp. 1059-1068 ◽

Cited By ~ 11

Author(s):

Thomas G. Bartholow ◽

Brianne L. Sanford ◽

Bach Cao ◽

Heidi L. Schmit ◽

James M. Johnson ◽

...

Keyword(s):

Trna Synthetase ◽

Editing Domain

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Structural Basis for Substrate Recognition by the Editing Domain of Isoleucyl-tRNA Synthetase

Journal of Molecular Biology ◽

10.1016/j.jmb.2006.04.025 ◽

2006 ◽

Vol 359 (4) ◽

pp. 901-912 ◽

Cited By ~ 38

Author(s):

Ryuya Fukunaga ◽

Shigeyuki Yokoyama

Keyword(s):

Substrate Recognition ◽

Trna Synthetase ◽

Structural Basis ◽

Editing Domain

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

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309112042066 ◽

2012 ◽

Vol 68 (11) ◽

pp. 1390-1393 ◽

Cited By ~ 1

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.

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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

Biochemical Journal ◽

10.1042/bj20051249 ◽

2006 ◽

Vol 394 (2) ◽

pp. 399-407 ◽

Cited By ~ 20

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.

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

Nucleic Acids Research ◽

10.1093/nar/gkab856 ◽

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.

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Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00873-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5817-5827 ◽

Cited By ~ 34

Author(s):

Andrés Palencia ◽

Ru-Juan Liu ◽

Maria Lukarska ◽

Jiri Gut ◽

Alexandre Bougdour ◽

...

Keyword(s):

Mammalian Cells ◽

Diarrheal Disease ◽

Trna Synthetase ◽

Content Type ◽

Apicomplexan Parasites ◽

Malnourished Children ◽

Covalent Adduct ◽

Editing Domain ◽

Antiparasitic Agents ◽

Micromolar Range

ABSTRACTThe apicomplexan parasitesCryptosporidiumandToxoplasmaare serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth ofCryptosporidiumparasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain ofCryptosporidiumleucyl-tRNA synthetase (LeuRS). The same compound was shown to be active againstToxoplasmaparasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in bothCryptosporidiumandToxoplasmaby forming a covalent adduct with tRNALeuin the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.

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