Tuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, Mycobacterium tuberculosis (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ provides the required reducing power for lipid biosynthesis. Here, we present the X-ray crystal structure of MEZ to 3.6 Å resolution and compare it with known structures of prokaryotic and eukaryotic malic enzymes. We use biochemical assays to determine its oligomeric state and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn²⁺ and NAD(P)⁺, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually disordered, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that MEZ subunits alternate between open and closed states and that MEZ can stably bind its NAD(P)⁺ cofactor in multiple conformations, including an inactive, compact NAD⁺ form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb.

中文翻译：

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抗结核药物潜在靶点结核分枝杆菌苹果酸酶的结构和分子动力学

结核病（TB）是全球最致命的细菌传染病。众所周知，这很难治疗，需要在数月内服用多种抗生素。结核分枝杆菌结核病菌的致密蜡状外膜（Mtb）是阻止抗生素吸收的强大屏障。随后，涉及维持Mtb细胞壁完整性的酶是有前途的药物靶标。最近，我们证明缺乏苹果酸酶（MEZ）的Mtb改变了细胞壁脂质的组成，并减少了巨噬细胞的摄取。这些结果表明MEZ提供了脂质生物合成所需的还原能力。在这里，我们介绍了MEZ的X射线晶体结构，分辨率为3.6Å，并将其与已知的原核和真核苹果酸酶结构进行了比较。我们使用生化分析来确定其低聚状态，并评估pH和变构调节剂对其动力学和热稳定性的影响。为了评估MEZ及其底物苹果酸和辅因子Mn ²⁺和NAD（P）之间的相互作用⁺，我们进行了一系列的分子动力学（MD）模拟。首先，MD分析证实了我们的经验性观察，即MEZ异常异常，即使加入底物和辅助因子，这种现象仍然存在。其次，MD模拟显示MEZ亚基在打开状态和关闭状态之间交替，并且MEZ可以多种构型稳定地结合其NAD（P）⁺辅因子，包括无活性，紧凑的NAD ⁺形式。MEZ的结构以及其动力学的见解可以一起用于设计靶向Mtb的MEZ抑制剂。