Abstract
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.
Highlights
Tuberculosis (TB), one of the oldest known human diseases, is still one of the leading infectious disease killers, despite the availability of drugs and the use of attenuated vaccines
Our findings suggest that changes in cellular cation concentrations contribute to phosphoenolpyruvate carboxykinase (Pck) catalysis and function in various stages of Mycobacterium tuberculosis (Mtb) infection
The key role of Pck in regulating the central carbon metabolism of Mtb has been confirmed, little is known about the properties of this enzyme
Summary
Tuberculosis (TB), one of the oldest known human diseases, is still one of the leading infectious disease killers, despite the availability of drugs and the use of attenuated vaccines. Mtb is not subjected to catabolic repression and is able to catabolize multiple carbon sources simultaneously (fatty acids, cholesterol, dextrose, acetate, glycerol, glucose) [3]. This ability likely helps Mtb adapt to the host niche. Numerous studies have shown that central carbon metabolism, which interconnects glycolysis, gluconeogenesis, and the tricarboxylic acid cycle, is altered in latent Mtb. The phosphoenolpyruvate-pyruvate-oxaloacetate node is critical for distribution of carbon flux through central metabolic pathways. Structural studies of GTP-dependent Pcks [11] indicated the presence of C- and N-terminal lobes, with the active site between the two lobes. The R- and P-loops are involved directly in substrate binding and catalysis, and the flexible Ω-loop lid domain, which undergoes an open-closed transition during substrate binding, stabilizes the correct position of the substrate for catalysis [8,9,12] and protects the enolate intermediate from protonation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
