Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

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.

The Role of Cysteine Residues in Catalysis of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

“…The distance measurement between all possible cysteine pairs in the X-ray crystal structure of reduced form of wt Pck in complex with GDP and Mn 2+ (PDB 4RCG; 4R43) [ 10 ] indicates four possible S-S bridge variants ( Fig 1A ) with Cys–Cys distances not exceeding 15.0 Å: C119–C198 (13.6 Å), C391–C49 (10.5 Å), C230–C89 (12.3 Å), and C391–C397 (14.4 Å). To confirm presence of disulfides, we performed LC-MS/MS analysis of pepsin digest of Pck sample without addition of reducing agent.…”

“…Cys residues are shown as sticks, and the potential S–S bridges are marked by dotted lines. The criterion for selection was a Cys–Cys distance less than 15.0 Å in the X-ray structure of reduced form of MTb Pck (PDB code: 4R43) [ 10 ]. (B) Position and orientation of C391, C397 in the X-ray structure of reduced wt Pck[ 10 ].…”

“…The prepared and optimized Pck-GDP-Mn 2+ complex from our previous work [ 10 ] was used. For this analysis, we removed the cofactor to match the mass spectrometry conditions.…”

“…For this analysis, we removed the cofactor to match the mass spectrometry conditions. Likewise, we discarded the Mn 2+ ion, which has a negligible effect on the structure [ 10 ], and the water molecules. A truncated octahedron of explicit TIP3P water molecules, which extended 11 Å from the protein, was added in the Leap module of AMBER14 [ 12 ].…”

“…The MTb Pck sequence contains nine cysteine residues, six of them (C49, C89, C119, C131, C198, and C230) are situated in the N-terminal domain (C89, C119, and C198 are located in random coil loops; C49 and C230 in α-helices, C131 in a β-sheet), C391 and C397 are present in the C-terminal subdomain between the N- and C-terminal domains. The reactive cysteine C273 that is typical for the GTP-dependent Pck family is localized in the binding site within the C-terminal domain, close to the binding site for GDP/GTP [ 10 ]. Mutational analysis, however, showed that C273 residue is not essential for Pck function in either directions [ 9 ].…”