Background: Multi drug-resistant tuberculosis is a major health threat to humans. Whole genome sequencing of several isoniazid (INH) resistant strains of M. tuberculosis revealed mutations in several genes. Rv1592c was demonstrated as lipolytic enzyme and its expression was up-regulated during isoniazid (INH) treatment. The valine at position 430 of Rv1592c was mutated to alanine frequently in the INH resistant strain of M. tuberculosis.

Methods: In this report, an array of computational approaches was used to understand the role of Val430-Ala mutation in Rv1592c in INH resistance. The impact of mutations on structural stability and degree of INH modification was demonstrated using the molecular dynamics method. The mutation in the Rv1592c gene at V430 position was created by the PCR primer walking method. Mutant and wild type gene was cloned into E. coli-mycobacteria shuttle vector (pVV-16) and expressed in Mycobacterium smegmatis system. The isoniazid susceptibility assay was performed by agar plate culture spot and CFUs count assay.

Results: This study demonstrated that the Val430 in Rv1592c makes the part of flap covering the substrate binding cavity. Mutation at Val430-Ala in Rv1592c caused the displacement of the flap region, resulting in uncovering a cavity, which allows accessibility of substrate to the active site cleft. The Val430-Ala mutation in Rv1592c created its structure energetically more stable. RMSD, RMSF and Rg simulation of mutant maintained overall stability throughout the simulation period while the native protein displayed comparatively more fluctuations. Moreover, docking studies showed that INH was bound into the active pocket of the mutant with considerable binding energy (−6.3 kcal/mol). In order to observe constant binding for INH, complexes were simulated for 50 ns. It was observed that after simulation, INH remained bound in the pocket with an increased molecular bonding network with the neighbor amino acid residues. In vitro studies clearly suggested that M. smegmatis expressing mutant has a better survival rate in isoniazid treatment as compared to wild type.

Conclusion: Overall, this study at the outset suggested that the mutation observed in drug resistant strain provides stability to the Rv1592c protein and increased affinity towards the INH due to flap displacement, leading to the possibility for its modification. In vitro results supported our in silico findings.

背景：耐多药结核病是对人类健康的主要威胁。对结核分枝杆菌的几种异烟肼 (INH) 耐药菌株的全基因组测序揭示了几种基因的突变。Rv1592c 被证明是脂肪分解酶，其表达在异烟肼 (INH) 处理期间上调。Rv1592c 430 位的缬氨酸在结核分枝杆菌的 INH 抗性菌株中经常突变为丙氨酸。

方法：在本报告中，使用一系列计算方法来了解 Rv1592c 中 Val430-Ala 突变在 INH 抗性中的作用。使用分子动力学方法证明了突变对结构稳定性和 INH 修饰程度的影响。V430 位置的 Rv1592c 基因突变是通过 PCR 引物步移法产生的。突变型和野生型基因被克隆到大肠杆菌-分枝杆菌穿梭载体（pVV-16）中，并在耻垢分枝杆菌系统中表达。异烟肼药敏试验采用琼脂平板培养点法和 CFUs 计数法。

结果：本研究表明，Rv1592c 中的 Val430 使皮瓣部分覆盖了基材结合腔。Rv1592c 中 Val430-Ala 的突变导致皮瓣区域的位移，导致空腔暴露，这使得底物可接近活性位点裂缝。Rv1592c 中的 Val430-Ala 突变使其结构在能量上更加稳定。突变体的 RMSD、RMSF 和 Rg 模拟在整个模拟期间保持整体稳定性，而天然蛋白质显示出相对更多的波动。此外，对接研究表明，INH 以相当大的结合能（-6.3 kcal/mol）结合到突变体的活性口袋中。为了观察 INH 的恒定结合，对复合物进行了 50 ns 的模拟。仿真后发现，INH 仍然结合在口袋中，与相邻氨基酸残基的分子结合网络增加。体外研究清楚地表明，与野生型相比，耻垢分枝杆菌表达突变体在异烟肼治疗中具有更好的存活率。

结论：总体而言，这项研究从一开始就表明在耐药菌株中观察到的突变为 Rv1592c 蛋白提供了稳定性，并由于瓣置换而增加了对 INH 的亲和力，从而导致对其进行修饰的可能性。体外结果支持我们的计算机研究结果。