Molecular Medicine - Structural Parasitology, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India.
Molecular Medicine - Structural Parasitology, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India; ICMR-National Institute of Malaria Research, Sector 8, Dwarka, Delhi 110077, India.
Mol Biochem Parasitol. 2022 Jul;250:111488. doi: 10.1016/j.molbiopara.2022.111488. Epub 2022 May 26.
The specificity of each aminoacyl-tRNA synthetase (aaRS) for its cognate amino acid ensures correct tRNA esterification and allows fidelity in protein synthesis. The aaRSs discriminate based on the chemical properties of their amino acid substrates and structural features of the binding pockets. In this study, we characterized aspartyl-(DRS) and asparaginyl-tRNA synthetase (NRS) from Plasmodium falciparum to determine the basis of their specificity towards L-asp and L-asn respectively. The negatively charged L-asp and its analogue L-asn differ only in their side-chain groups i.e., -OH and -NH. Further, the amino acid binding sites are highly conserved within these two enzymes. Analysis of the substrate (L-asp/L-asn) binding sites across species revealed two highly conserved residues in PfDRS (D408 and K372) and PfNRS (E395 and L360) that are involved in recognition of the O/N of L-asp/L-asn respectively. These residues were mutated and swapped between the D408→E in PfDRS and the corresponding E395→D in PfNRS. A similar approach was employed for residue number K372→L in PfDRS and L360→K in PfNRS. The mutated PfDRS retained its enzymatic activity during step 1 of aminoacylation reaction towards L-asp and L-asn and esterified tRNA with L-asp like wild type enzyme, while the PfDRS was rendered enzymatically inactive. The correspondingly mutated PfNRS was enzymatically inactive. The mutated PfNRS had an altered specificity and esterified tRNA with non-cognate amino acid L-asp and not L-asn. These data suggest that the residue K372 is crucial for the enzymatic activity of PfDRS while the residue L360 in PfNRS imparts specificity towards L-asn.
每种氨酰-tRNA 合成酶（aaRS）对其同源氨基酸的特异性确保了正确的 tRNA 酯化，并允许蛋白质合成具有保真度。aaRS 基于其氨基酸底物的化学性质和结合口袋的结构特征进行区分。在这项研究中，我们对恶性疟原虫的天冬氨酰-tRNA 合成酶（DRS）和天冬酰胺酰-tRNA 合成酶（NRS）进行了表征，以确定它们分别对 L-天冬氨酸和 L-天冬酰胺的特异性基础。带负电荷的 L-天冬氨酸及其类似物 L-天冬酰胺仅在其侧链基团即-OH 和-NH 上有所不同。此外，这两种酶的氨基酸结合位点高度保守。对跨物种的底物（L-天冬氨酸/L-天冬酰胺）结合位点的分析表明，PfDRS（D408 和 K372）和 PfNRS（E395 和 L360）中存在两个高度保守的残基，它们分别参与 L-天冬氨酸/L-天冬酰胺的 O/N 的识别。这些残基在 PfDRS 中从 D408→E 和 PfNRS 中相应的 E395→D 之间发生突变和交换。同样的方法也用于 PfDRS 中的残基 K372→L 和 PfNRS 中的 L360→K。突变的 PfDRS 在 L-天冬氨酸和 L-天冬酰胺的氨酰化反应的第一步中保留了其酶活性，并像野生型酶一样将 tRNA 与 L-天冬氨酸酯化，而 PfDRS 则失去了酶活性。相应突变的 PfNRS 没有酶活性。突变的 PfNRS 具有改变的特异性，并将非同源氨基酸 L-天冬氨酸而不是 L-天冬酰胺酯化 tRNA。这些数据表明，残基 K372 对 PfDRS 的酶活性至关重要，而 PfNRS 中的残基 L360 赋予其对 L-天冬酰胺的特异性。
