Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.

• MeSH
• aminokyselinové motivy MeSH
• biokatalýza * MeSH
• cystein metabolismus   MeSH
• disulfidy metabolismus   MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) chemie   metabolismus   MeSH
• kinetika MeSH
• molekulární modely MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• mutantní proteiny chemie   MeSH
• Mycobacterium tuberculosis enzymologie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• tandemová hmotnostní spektrometrie MeSH
• terciární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• disulfidy MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) MeSH
• mutantní proteiny MeSH

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.

• Klíčová slova
• Enzyme Kinetics, Hypoxia, Metabolism, Mycobacterium Tuberculosis, Oxidation-Reduction, Phosphoenolpyruvate Carboxykinase, Thioredoxin,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• citrátový cyklus fyziologie   MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) genetika   metabolismus   MeSH
• Mycobacterium tuberculosis enzymologie   genetika   MeSH
• oxidace-redukce MeSH
• regulace genové exprese enzymů fyziologie   MeSH
• regulace genové exprese u bakterií fyziologie   MeSH
• thioredoxiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) MeSH
• thioredoxiny MeSH