From the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague, Czech Republic.
J Biol Chem. 2014 May 9;289(19):13066-78. doi: 10.1074/jbc.M113.536748. Epub 2014 Mar 21.
Tuberculosis remains a major health concern worldwide. Eradication of its causative agent, the bacterial pathogen Mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. Despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. Hypoxic conditions have been widely used as an in vitro system to study this dormancy. Such studies identified a rearrangement of central carbon metabolism to exploit fermentative processes caused by the lack of oxygen. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is the enzyme at the center of these metabolic rearrangements. Although Pck is associated with gluconeogenesis under standard growth conditions, the enzyme can catalyze the reverse reaction, supporting anaplerosis of the tricarboxylic acid cycle, under conditions leading to slowed or stopped bacterial replication. To study the mechanisms that regulate the switch between two Pck functions, we systematically investigated factors influencing the gluconeogenic and anaplerotic reaction kinetics. We demonstrate that a reducing environment, as found under hypoxia-triggered non-replicating conditions, accelerates the reaction in the anaplerotic direction. Furthermore, we identified proteins that interact with Pck. The interaction between Pck and the reduced form of mycobacterial thioredoxin, gene expression of which is increased under hypoxic conditions, also increased the Pck anaplerotic activity. We thus propose that a reducing environment and the protein-protein interaction with thioredoxin in particular enable the Pck anaplerotic function under fermentative growth conditions.
结核病仍然是全球主要的健康问题。由于潜伏性疾病的大量携带者，消除其病原体结核分枝杆菌尤其具有挑战性。尽管与潜伏感染相关的所谓休眠状态的术语具有误导性，但细菌必须维持基本的代谢活动。缺氧条件已被广泛用作研究这种休眠状态的体外系统。这些研究确定了中心碳代谢的重排，以利用由于缺氧引起的发酵过程。磷酸烯醇丙酮酸羧激酶（Pck；EC 4.1.1.32）是这些代谢重排的中心酶。虽然 Pck 在标准生长条件下与糖异生有关，但在导致细菌复制减缓或停止的条件下，该酶可以催化反向反应，支持三羧酸循环的回补作用。为了研究调节两种 Pck 功能之间转换的机制，我们系统地研究了影响糖异生和回补反应动力学的因素。我们证明，还原环境（如缺氧触发的非复制条件下存在的环境）会加速回补方向的反应。此外，我们鉴定了与 Pck 相互作用的蛋白质。Pck 与还原型分枝杆菌硫氧还蛋白的相互作用，其基因表达在缺氧条件下增加，也增加了 Pck 的回补活性。因此，我们提出还原环境和与硫氧还蛋白的蛋白质-蛋白质相互作用特别使 Pck 能够在发酵生长条件下发挥回补功能。
