Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

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

Helical supramolecular assemblies of {2,4,6-[Cp*Rh(E2-1,2-C2B10H10)(NC5H4CH2S)]3(triazine)} (E = S, Se) shaped by Cp*-toluene-Cp* .pi.-stacking forces and BH-pyridine hydrogen bonding

Dendritic half-sandwich rhodium carborane trimers, [Cp*Rh(1,2-E2-1,2-C2B10H10)]3(tpst) [2a,b; Cp* = .eta.5-C5Me5, tpst = 2,4,6-tris(4-pyridinyl-.kappa.N)methylthio-1,3,5-triazine, E = S, Se] were prepd. by complexation of [Cp*Rh(1,2-E2-1,2-C2B10H10)] with tpst ligand. The complexes 2a,b form toluene solvates in the solid state showing infinitely connected [(2a,b)-(toluene)] helixes. The chains of these supramols. are held together by Cp*-toluene-Cp* .pi.-stacking interactions of two of the three Cp* ligands of the bell-shaped 2a and 2b mols. Unconventional BH.delta.--pyridyl.delta.+ arom. hydrogen bonding enforces the bell-shapes of the mol. units, and the Cp* conformations are expected to induce the supramol. structures