Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad 500046, Telangana, India.
Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad 500046, Telangana, India.
Int J Biol Macromol. 2020 Dec 15;165(Pt B):2869-2885. doi: 10.1016/j.ijbiomac.2020.10.140. Epub 2020 Oct 22.
Aminoacyl tRNA synthetases (aaRSs) are integral components of protein biosynthesis along with several non-canonical cellular processes. Inhibition studies of aaRSs presented these enzymes as promising drug targets in many pathogens, however aspartyl tRNA synthetase has not been studied in trypanosomatids despite its essentiality. Hence, full-length ORF of Leishmania donovani aspartyl tRNA synthetase (LdaspRS) was cloned and purified to homogeneity followed by molecular mass determination. The aminoacylation assay established that the purified protein performs its function optimally at physiological pH and temperature. The kinetic parameters of LdaspRS revealed the affinity of l-aspartate towards the enzyme to be very much lower than the cofactor. Our study also highlights the moonlighting function of LdaspRS to stimulate the pro-inflammatory cytokines and nitric oxide generation by host macrophage. Furthermore, CD and intrinsic tryptophan fluorescence measurements showed the changes in structural conformation at varying pH, denaturants and ligands. The modelled LdaspRS structure presented all the specific characteristics of class II aaRSs, while in silico study suggested binding of pyrimidine-derived inhibitors in its cofactor binding site with high affinity followed by validation using MD simulation. Altogether, this study could provide a platform for exploring LdaspRS to develop potential therapeutics against leishmaniasis.
氨酰-tRNA 合成酶（aaRSs）是蛋白质生物合成以及几种非典型细胞过程的重要组成部分。aaRSs 的抑制研究表明，这些酶是许多病原体中有希望的药物靶点，然而，尽管天冬酰-tRNA 合成酶是必需的，但在原生动物中尚未对其进行研究。因此，克隆并纯化了完整的 Leishmania donovani 天冬酰-tRNA 合成酶（LdaspRS）全长 ORF，并使其达到均一性，随后确定了其分子量。氨酰化测定确定，纯化的蛋白质在生理 pH 值和温度下最佳地发挥其功能。LdaspRS 的动力学参数表明，天冬氨酸对酶的亲和力远低于辅因子。我们的研究还强调了 LdaspRS 的兼职功能，可刺激宿主巨噬细胞中促炎细胞因子和一氧化氮的产生。此外，圆二色性（CD）和固有色氨酸荧光测量显示了在不同 pH 值、变性剂和配体下结构构象的变化。所构建的 LdaspRS 结构呈现出所有 II 类 aaRSs 的特异性特征，而计算机模拟研究表明，嘧啶衍生抑制剂可以与辅因子结合位点高亲和力结合，随后通过 MD 模拟进行验证。总的来说，这项研究为探索 LdaspRS 以开发针对利什曼病的潜在治疗方法提供了一个平台。
