A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation

• Published in: Biochimica et biophysica acta Vol. 1850; no. 1; pp. 43 - 50
• Main Authors: Auger, Christopher, Appanna, Vasu D.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2015
• Subjects: Adaptation, Physiological; adenosine triphosphate; Adenosine Triphosphate - metabolism; adenylate kinase; Adenylate Kinase - metabolism; anti-infective agents; bacteria; Bacterial Proteins - metabolism; Chromatography, High Pressure Liquid; citrates; Citric Acid Cycle; Electrophoresis, Polyacrylamide Gel; energy; high performance liquid chromatography; macrophages; Metabolic Networks and Pathways; Metabolism; Metabolon; Microbiology; Models, Biological; molecular conformation; Multienzyme Complexes - metabolism; nitric oxide; Nitric Oxide - metabolism; nitrogen; nucleoside-diphosphate kinase; Nucleoside-Diphosphate Kinase - metabolism; Oxidative Phosphorylation; Oxo-Acid-Lyases - metabolism; pathogens; phosphoenolpyruvate carboxylase; Phosphoenolpyruvate Carboxylase - metabolism; Phosphotransfer; polyacrylamide gel electrophoresis; Pseudomonas; Pseudomonas fluorescens - metabolism; pyruvate phosphate dikinase; Pyruvate, Orthophosphate Dikinase - metabolism; Reactive nitrogen species; Reactive Nitrogen Species - metabolism; Stress, Physiological; tricarboxylic acid cycle; NO; CFE; Microbiology; Metabolon; Reactive nitrogen species; ADP; PEPC; SNP; NDPK; RNS; SLP; Phosphotransfer; HPLC; PPDK; AMP; BN-PAGE; G6PDH; ACK; CL; AK; Metabolism; TCA; ETC; PEP; PK; ATP; DEANO
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2014.09.028

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 O2-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. 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. 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. 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. 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. [Display omitted] •Nitric oxide exposure hinders the electron transport chain.•An alternative means of generating ATP is evoked in Pseudomonas fluorescens.•Citrate lyase, phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase are linked.•This multi-enzymatic complex is conducive to microbial survival.