Abstract：
Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.
摘要：
氨酰tRNA合成酶（aaRS）是蛋白质翻译机制的基本组成部分。鉴于它们在蛋白质合成和物种间结构差异中的关键作用，它们一直被认为是开发抗菌化合物的潜在目标。来自克氏锥虫的精氨酰tRNA合成酶（TcArgRS），导致查加斯病的寄生虫,含有100个氨基酸的插入，在相似长度的人类对应物中完全不存在，如从多个序列比对结果确定的。因此，我们被提示使用生物物理技术对TcArgRS进行初步表征，生物化学，和生物信息学工具。我们在大肠杆菌中表达了该蛋白，并验证了其体外酶活性。此外,DTNB动力学分析，圆形二向色（CD）光谱，和配体结合研究使用固有色氨酸荧光测量帮助我们了解一些结构特征在缺乏可用的晶体结构。我们的研究表明，TcArgRS可以区分L-精氨酸及其类似物。在许多测试的基底中，只有L-刀豆氨酸和L-硫代精氨酸，合成的精氨酸类似物表现出显著的活化。还使用计算机模拟方法确定了各种底物的结合。这项研究可能为研究可靶向TcArgRS的小化合物提供了可行的基础。
