A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation
A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation
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DOI：
10.1016/j.bbagen.2014.09.028
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发表时间：
2015-01-01
影响因子：
3
通讯作者：
Appanna, Vasu D.
中科院分区：
文献类型：
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作者：
Auger, Christopher;Appanna, Vasu D.
背景：众所周知，一氧化氮和其他活性氮物质 (RNS) 含量的增加会对三羧酸 (TCA) 循环和氧化磷酸化产生负面影响。这些扰动严重损害了依赖 O-2 的能源生产。虽然已知细菌能够适应 RNS（巨噬细胞对抗感染的关键工具），但确切的机制尚不清楚。方法：将细菌在特定的矿物质培养基中培养，并利用同一生长期获得的无细胞提取物进行各种生化研究。应用蓝色天然聚丙烯酰胺凝胶电泳，然后进行凝胶内活性测定、高效液相色谱和免疫共沉淀来研究 RNS 对模型微生物假单胞菌的影响结果：使用由柠檬酸裂解酶 (CL)、磷酸烯醇丙酮酸羧化酶 (PEPC) 和丙酮酸磷酸二激酶 (PPDK) 组成的新型代谢物将柠檬酸从三羧酸循环中引导出来。这种由三种不同酶组成的代谢引擎似乎短暂地组装成一个旨在合成 ATP 的超级复合物。腺苷酸激酶 (AK) 和核苷二磷酸激酶 (NDPK) 活性的上调确保了这种 ATP 制造机器的功效。结论：微生物可以通过重新设计代谢网络来逃避亚硝化应激的影响，以便在电子传递链缺陷时厌氧地生成和储存 ATP。一般意义：本文描述的分子配置提供了对代谢如何在适应亚硝化应激中发挥关键作用的进一步理解，并揭示了新的靶点这将为对抗 RNS 耐药病原体的抗菌药物的开发提供信息。 (C) 2014 Elsevier B.V. 保留所有权利。
Background: It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O-2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown.Methods: The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens.Results: Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine.Conclusion: Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective.General significance: The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens. (C) 2014 Elsevier B.V. All rights reserved.