From the Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH.
the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, and.
J Biol Chem. 2018 Apr 13;293(15):5695-5704. doi: 10.1074/jbc.RA118.001839. Epub 2018 Feb 23.
Enzymes at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, (). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of We show that in addition to PCK, intracellular requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO through the ANA node is essential for intracellular survival of and that possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.
磷酸烯醇丙酮酸（PEP）-丙酮酸-草酰乙酸或补充途径（ANA）节点的酶控制着糖酵解、糖异生和补充途径的代谢通量。在这里，我们使用遗传、生化和 C 同位素分析来表征 ANA 节点中的酶在世界上最成功的细菌病原体 （）细胞内生存中的作用。我们表明，四个 ANA 酶中的每一个，即丙酮酸羧化酶（PCA）、PEP 羧激酶（PCK）、苹果酸酶（MEZ）和丙酮酸磷酸二激酶（PPDK），在 细胞内生存中都具有独特而必需的代谢功能。我们表明，除了 PCK 之外，细胞内 还需要 PPDK 作为进入糖异生的替代途径。丙酸和胆固醇解毒也被确定为 PPDK 的必需功能，这揭示了 ANA 节点在这些生理上重要的细胞内底物代谢中的意外作用，并强调了该酶作为结核病（TB）特异性药物靶点的作用。我们表明，通过 ANA 节点固定 CO2 对于 细胞内生存是必需的，并且 拥有三种能够完成此功能的酶（PCA、PCK 和 MEZ）。除了在补充途径中提供备份作用外，我们还表明 MEZ 也在脂质生物合成中具有作用。MEZ 敲除株具有改变的细胞壁并且在最初进入巨噬细胞时缺乏。这项工作揭示了 ANA 节点是控制 细胞内复制的焦点，这超出了经典的糖异生作用，代表了设计新型抗结核药物的有希望的靶点。
