Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia.
Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, 4041, South Africa.
Sci Rep. 2020 Nov 3;10(1):18925. doi: 10.1038/s41598-020-75799-8.
Pulmonary tuberculosis, caused by Mycobacterium tuberculosis, is one of the most persistent diseases leading to death in humans. As one of the key targets during the latent/dormant stage of M. tuberculosis, isocitrate lyase (ICL) has been a subject of interest for new tuberculosis therapeutics. In this work, the cleavage of the isocitrate by M. tuberculosis ICL was studied using quantum mechanics/molecular mechanics method at M06-2X/6-31+G(d,p): AMBER level of theory. The electronic embedding approach was applied to provide a better depiction of electrostatic interactions between MM and QM regions. Two possible pathways (pathway I that involves Asp108 and pathway II that involves Glu182) that could lead to the metabolism of isocitrate was studied in this study. The results suggested that the core residues involved in isocitrate catalytic cleavage mechanism are Asp108, Cys191 and Arg228. A water molecule bonded to Mg acts as the catalytic base for the deprotonation of isocitrate C(2)-OH group, while Cys191 acts as the catalytic acid. Our observation suggests that the shuttle proton from isocitrate hydroxyl group C(2) atom is favourably transferred to Asp108 instead of Glu182 with a lower activation energy of 6.2 kcal/mol. Natural bond analysis also demonstrated that pathway I involving the transfer of proton to Asp108 has a higher intermolecular interaction and charge transfer that were associated with higher stabilization energy. The QM/MM transition state stepwise catalytic mechanism of ICL agrees with the in vitro enzymatic assay whereby Asp108Ala and Cys191Ser ICL mutants lost their isocitrate cleavage activities.
肺结核，由结核分枝杆菌引起，是导致人类死亡的最持久疾病之一。异柠檬酸裂解酶（ICL）作为结核分枝杆菌潜伏/休眠阶段的关键靶标之一，一直是新型抗结核治疗的研究对象。在这项工作中，使用量子力学/分子力学方法在 M06-2X/6-31+G(d,p)：AMBER 理论水平上研究了结核分枝杆菌 ICL 对异柠檬酸的裂解。应用电子嵌入方法可以更好地描述 MM 和 QM 区域之间的静电相互作用。本研究研究了两条可能的途径（涉及 Asp108 的途径 I 和涉及 Glu182 的途径 II），这些途径可能导致异柠檬酸的代谢。结果表明，参与异柠檬酸催化裂解机制的核心残基是 Asp108、Cys191 和 Arg228。与 Mg 键合的水分子作为异柠檬酸 C(2)-OH 基团去质子化的催化碱，而 Cys191 作为催化酸。我们的观察表明，来自异柠檬酸羟基 C(2)原子的穿梭质子更有利于转移到 Asp108，而不是 Glu182，其活化能低至 6.2 kcal/mol。自然键分析还表明，涉及质子转移到 Asp108 的途径 I 具有更高的分子间相互作用和电荷转移，与更高的稳定化能相关。ICL 的 QM/MM 过渡态逐步催化机制与体外酶测定一致，其中 Asp108Ala 和 Cys191Ser ICL 突变体失去了异柠檬酸裂解活性。
