Khan Muhammad Tahir, Junaid Muhammad, Mao Xueying, Wang Yanjie, Hussain Abid, Malik Shaukat Iqbal, Wei Dong-Qing
Department of Bioinformatics and Biosciences, Capital University of Science and Technology, Islamabad, Pakistan.
Department of Bioinformatics and Biostatistics, College of Life Sciences and Biotechnology, The State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, China.
J Cell Biochem. 2019 May;120(5):7154-7166. doi: 10.1002/jcb.27989. Epub 2018 Nov 28.
Pyrazinamide (PZA) is an important component of first-line antituberculosis drugs activated by Mycobacterium tuberculosis pyrazinamidase (PZase) into its active form pyrazinoic acid. Mutations in the pncA gene have been recognized as the major cause of PZA resistance. We detected some novel mutations, Leucine19Arginine (L19R), Arginine140Histidine (R140H), and Glutamic acid144 Lysine (E144K), in the pncA gene of PZA-resistant isolates in our wet lab PZA drug susceptibility testing and sequencing. As the molecular mechanism of resistance of these variants has not been reported earlier, we have performed multiple analyses to unveil different mechanisms of resistance because of PZase mutations L19R, R140H, and E144K. The mutants and native PZase structures were subjected to comprehensive computational molecular dynamics (MD) simulations at 100 nanoseconds in apo and drug-bound form. Mutants and native PZase binding pocket were compared to observe the consequence of mutations on the binding pocket size. Hydrogen bonding, Gibbs free energy, and natural ligand Fe effect were also analyzed between native and mutants. A significant variation between native and mutant PZase structure activity was observed. The native PZase protein docking score was found to be the maximum, showing strong binding affinity in comparison with mutants. MD simulations explored the effect of the variants on the biological function of PZase. Hydrogen bonding, metal ion Fe deviation, and fluctuation also seemed to be affected because of the mutations L19R, R140H, and E144K. The variants L19R, R140H, and E144K play a significant role in PZA resistance, altering the overall activity of native PZase, including metal ion Fe displacement and free energy. This study offers valuable evidence for better management of drug-resistant tuberculosis.
吡嗪酰胺（PZA）是一线抗结核药物的重要组成部分，它被结核分枝杆菌吡嗪酰胺酶（PZase）激活成其活性形式吡嗪酸。pncA基因突变已被认为是PZA耐药的主要原因。在我们的湿实验室PZA药物敏感性测试和测序中，我们在耐PZA分离株的pncA基因中检测到了一些新的突变，即亮氨酸19突变为精氨酸（L19R）、精氨酸140突变为组氨酸（R140H）和谷氨酸144突变为赖氨酸（E144K）。由于这些变体的耐药分子机制此前尚未见报道，我们进行了多项分析，以揭示由PZase突变L19R、R140H和E144K导致的不同耐药机制。对突变体和天然PZase结构在无配体和药物结合形式下进行了100纳秒的全面计算分子动力学（MD）模拟。比较了突变体和天然PZase的结合口袋，以观察突变对结合口袋大小的影响。还分析了天然型和突变体之间的氢键、吉布斯自由能和天然配体Fe效应。观察到天然型和突变型PZase结构活性之间存在显著差异。发现天然PZase蛋白对接分数最高，与突变体相比显示出很强的结合亲和力。MD模拟探索了这些变体对PZase生物学功能的影响。由于突变L19R、R140H和E144K，氢键、金属离子Fe偏差和波动似乎也受到了影响。变体L19R、R140H和E144K在PZA耐药中起重要作用，改变了天然PZase的整体活性，包括金属离子Fe的位移和自由能。本研究为更好地管理耐多药结核病提供了有价值的证据。
