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Mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin.

ABSTRACT: 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.

SUBMITTER: Machova I

PROVIDER: S-EPMC4036320 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin.

Machová Iva I Snašel Jan J Zimmermann Michael M Laubitz Daniel D Plocinski Przemyslaw P Oehlmann Wulf W Singh Mahavir M Dostál Jiři J Sauer Uwe U Pichová Iva I

The Journal of biological chemistry 20140321 19

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 r ...[more]

PMID: 24659783

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Project description: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.

Project description:The bacterial pathogen Mycobacterium tuberculosis is the leading cause of death by an infectious disease among humans. Here, we describe a previously uncharacterized M. tuberculosis protein, Rv0991c, as a molecular chaperone that is activated by oxidation. Rv0991c has homologs in most bacterial lineages and appears to function analogously to the well-characterized Escherichia coli redox-regulated chaperone Hsp33, despite a dissimilar protein sequence. Rv0991c is transcriptionally coregulated with hsp60 and hsp70 chaperone genes in M. tuberculosis, suggesting that Rv0991c functions with these chaperones in maintaining protein quality control. Supporting this hypothesis, we found that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded protein in vitro and promotes its refolding by the M. tuberculosis Hsp70 chaperone system. Furthermore, Rv0991c interacts with DnaK and can associate with many other M. tuberculosis proteins. We therefore propose that Rv0991c, which we named "Ruc" (redox-regulated protein with unstructured C terminus), represents a founding member of a new chaperone family that protects M. tuberculosis and other species from proteotoxicity during oxidative stress.IMPORTANCE M. tuberculosis infections are responsible for more than 1 million deaths per year. Developing effective strategies to combat this disease requires a greater understanding of M. tuberculosis biology. As in all cells, protein quality control is essential for the viability of M. tuberculosis, which likely faces proteotoxic stress within a host. Here, we identify an M. tuberculosis protein, Ruc, that gains chaperone activity upon oxidation. Ruc represents a previously unrecognized family of redox-regulated chaperones found throughout the bacterial superkingdom. Additionally, we found that oxidized Ruc promotes the protein-folding activity of the essential M. tuberculosis Hsp70 chaperone system. This work contributes to a growing body of evidence that oxidative stress provides a particular strain on cellular protein stability.

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Mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin.

Publications

Mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin.

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