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Unlike the arginyl\u2010tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of <jats:italic>Leishmania donovani<\/jats:italic> (<jats:italic>Ld<\/jats:italic>ArgRS) that can follow the canonical two\u2010step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE\u2013PCR) method to create a deletion mutant (\u0394Ins\u2010<jats:italic>Ld<\/jats:italic>ArgRS) devoid of this region to investigate its function. Notably, the purified <jats:italic>Ld<\/jats:italic>ArgRS and \u0394Ins\u2010<jats:italic>Ld<\/jats:italic>ArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full\u2010length protein showed better catalytic efficiency than \u0394Ins\u2010<jats:italic>Ld<\/jats:italic>ArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo\u2010coumarin derivative (Comp\u20107j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of <jats:italic>Ld<\/jats:italic>ArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for \u0394Ins\u2010<jats:italic>Ld<\/jats:italic>ArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid\u2010specific insertion and Rossmann fold motif, <jats:italic>Ld<\/jats:italic>ArgRS revealed typical structural characteristics of ArgRSs, and Comp\u20107j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNA<jats:sup>Arg<\/jats:sup> near the insertion region enhanced the stability and compactness of <jats:italic>Ld<\/jats:italic>ArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti\u2010leishmanial agents.<\/jats:p>","DOI":"10.1111\/febs.17122","type":"journal-article","created":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T09:05:21Z","timestamp":1711357521000},"page":"2955-2979","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl\u2010tRNA synthetase"],"prefix":"10.1111","volume":"291","author":[{"given":"Fouzia","family":"Nasim","sequence":"first","affiliation":[{"name":"Department of Biotechnology &amp; Bioinformatics, School of Life Sciences University of Hyderabad  Hyderabad India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muppidi 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