Machová Iva, Snášel Jan, Dostál Jiří, Brynda Jiří, Fanfrlík Jindřich, Singh Mahavir, Tarábek Ján, Vaněk Ondřej, Bednárová Lucie, Pichová Iva
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
LIONEX diagnostics & Therapeutics, Braunschweig, Germany.
PLoS One. 2015 Mar 23;10(3):e0120682. doi: 10.1371/journal.pone.0120682. eCollection 2015.
Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.
结核病是仅次于艾滋病毒的第二大致命传染病杀手，仍然是公共卫生的首要重点。结核病的病原体结核分枝杆菌（Mtb）可引起急性感染和临床潜伏感染，它会根据宿主生态位重新编程新陈代谢。磷酸烯醇式丙酮酸羧激酶（Pck）是磷酸烯醇式丙酮酸-丙酮酸-草酰乙酸节点的核心酶，该节点参与调节结核分枝杆菌感染不同状态下碳流向分解代谢、合成代谢或呼吸作用的分布。在标准生长条件下，结核分枝杆菌Pck与糖异生作用相关，并催化磷酸烯醇式丙酮酸的金属依赖性形成。在非复制性结核分枝杆菌中，Pck可催化草酰乙酸的回补生物合成。在此，我们阐述了二价阳离子对结核分枝杆菌Pck活性的调节作用。通过对Pck-GDP和Pck-GDP-Mn2+复合物的X射线结构分析、GDP结合位点的突变分析以及基于量子力学（QM）的分析，我们探索了结核分枝杆菌Pck高效催化的结构决定因素。我们证明，结核分枝杆菌Pck需要Mn2+和Mg2+阳离子的存在才能高效催化糖异生和回补反应。回补反应在结核分枝杆菌复制减慢或停止的特征性还原条件下发挥作用，在Mn2+或Mg2+阳离子存在时，Fe2+也能有效激活该反应。相比之下，Fe2+与Mn2+或Mg2+同时存在会抑制糖异生反应。这些结果表明，无机离子可通过影响Pck的活性来调节中心碳代谢。此外，Pck突变体的X射线结构测定、生化特性分析和QM分析证实了苯丙氨酸三联体在核苷酸结合位点中对GDP-Mn2+复合物的正确结合以及回补反应的高效催化中的重要作用。
