School of Biochemical Engineering, Indian Institute of Technology BHU, Varanasi, UP 221005, India.
School of Biochemical Engineering, Indian Institute of Technology BHU, Varanasi, UP 221005, India.
Int J Biol Macromol. 2024 Nov;279(Pt 3):135400. doi: 10.1016/j.ijbiomac.2024.135400. Epub 2024 Sep 6.
Citrate synthase is a crucial enzyme in the TCA cycle and represents a potential therapeutic target. However, knowledge about this enzyme in Leishmania parasites remains limited. In this study, we have successfully cloned, expressed, and purified citrate synthase from Leishmania donovani (LdCS) using a bacterial system, and characterized it through various biophysical and biochemical methods. Circular dichroism analysis at physiological pH indicates that LdCS is properly folded. Further investigation into its tertiary structure using a quencher reveals that most tryptophan residues are located within the protein's hydrophobic core. Biochemical assays show that the recombinant enzyme is catalytically active, with optimal activity at pH 7.0. Kinetic studies provided parameters such as Km and Vmax. Enzyme inhibition assays revealed that LdCS activity is competitively inhibited by FDA-approved compounds-Abemaciclib, Bazedoxifene, Vorapaxar, and Imatinib-with Ki values ranging from 2 to 3 μM, demonstrating significant binding affinity. This research paves the way for exploring LdCS as a potential drug target for treating leishmaniasis.
柠檬酸合酶是三羧酸循环中的关键酶，是一个潜在的治疗靶点。然而，关于利什曼原虫中的这种酶的知识仍然有限。在这项研究中，我们使用细菌系统成功地从杜氏利什曼原虫（LdCS）中克隆、表达和纯化了柠檬酸合酶，并通过各种生物物理和生化方法对其进行了表征。生理 pH 值下的圆二色性分析表明 LdCS 正确折叠。进一步使用淬灭剂研究其三级结构表明，大多数色氨酸残基位于蛋白质的疏水核心内。生化测定表明，重组酶具有催化活性，在 pH 7.0 时具有最佳活性。动力学研究提供了 Km 和 Vmax 等参数。酶抑制测定表明，LdCS 活性被 FDA 批准的化合物-阿贝西利、巴多昔芬、沃拉帕沙和伊马替尼竞争性抑制，Ki 值范围为 2 至 3 μM，表明具有显著的结合亲和力。这项研究为探索 LdCS 作为治疗利什曼病的潜在药物靶点铺平了道路。
