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.

Department of Biochemistry Faculty of Sciences Charles University Prague Prague Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague Czech Republic

LIONEX diagnostics and Therapeutics Braunschweig Germany

Zobrazit více v PubMed

Gandhi NR, Nunn P, Dheda K, Schaaf HS, Zignol M, van Soolingen D, et al. Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis. Lancet. Elsevier Ltd; 2010;375:1830–43. 10.1016/S0140-6736(10)60410-2 PubMed DOI

Velayati AA, Masjedi MR, Farnia P, Tabarsi P, Ghanavi J, Ziazarifi AH, et al. Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in iran. Chest. 2009;136:420–5. 10.1378/chest.08-2427 PubMed DOI

De Carvalho LPS, Fischer SM, Marrero J, Nathan C, Ehrt S, Rhee KY. Metabolomics of Mycobacterium tuberculosis reveals compartmentalized co-catabolism of carbon substrates. Chem Biol. Elsevier Ltd; 2010;17:1122–31. 10.1016/j.chembiol.2010.08.009 PubMed DOI

Mukhopadhyay B, Concar EM, Wolfe RS. A GTP-dependent vertebrate-type phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis . J Biol Chem. 2001;276:16137–45. 10.1074/jbc.M008960200 PubMed DOI

Matte a, Tari LW, Goldie H, Delbaere LT. Structure and mechanism of phosphoenolpyruvate carboxykinase. J Biol Chem. 1997;272:8105–8. PubMed

Schöcke L, Weimer PJ. Purification and characterization of phosphoenolpyruvate carboxykinase from the anaerobic ruminal bacterium Ruminococcus flavefaciens . Arch Microbiol. 1997;167:289–94. PubMed

Sullivan SM, Holyoak T. Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid. Biochemistry. 2007;46:10078–88. 10.1021/bi701038x PubMed DOI

Holyoak T, Sullivan S, Nowak T. Structural insights into the mechanism of PEPCK catalysis. Biochemistry. 2006;45:8254–63. 10.1021/bi060269g PubMed DOI

Dunten P, Belunis C, Crowther R, Hollfelder K, Kammlott U, Levin W, et al. Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site. J Mol Biol. 2002;316:257–64. 10.1006/jmbi.2001.5364 PubMed DOI

Tortora P, Hanozet GM, Guerritore A. Purification of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae and its use for bicarbonate assay. Anal Biochem. 1985;144:179–185. 10.1016/0003-2697(85)90101-0 PubMed DOI

Carlson G, Holyoak T. Structural insights into the mechanism of phosphoenolpyruvate carboxykinase catalysis. J Biol Chem. 2009;284:27037–41. 10.1074/jbc.R109.040568 PubMed DOI PMC

Johnson T a, Holyoak T. The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function. Biochemistry. 2012;51:9547–59. 10.1021/bi301278t PubMed DOI PMC

Colombo G, Carlson GM, Lardy H a. Phosphoenolpyruvate carboxykinase (guanosine 5’-triphosphate) from rat liver cytosol. Dual-cation requirement for the carboxylation reaction. Biochemistry. 1981;20:2749–57. PubMed

Lee MH, Hebda CA, Nowak T. The role of cations in avian liver phosphoenolpyruvate carboxykinase catalysis. Activation and regulation. J Biol Chem. 1981;256:12793–801. PubMed

Hebdas, Candacia A NT. Phospho-enolpyruvate Carboxykinase.Mn2+ and Mn2+ substrate complexes. JBC. 1982;257:5515–5522. PubMed

Machová I, Snašel J, Zimmermann M, Laubitz D, Plocinski P, Oehlmann W, et al. Mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin. J Biol Chem. 2014;289:13066–78. 10.1074/jbc.M113.536748 PubMed DOI PMC

Beste DJ V, Nöh K, Niedenführ S, Mendum TA, Hawkins ND, Ward JL, et al. 13C-flux spectral analysis of host-pathogen metabolism reveals a mixed diet for intracellular Mycobacterium tuberculosis . Chem Biol. 2013;20:1012–21. 10.1016/j.chembiol.2013.06.012 PubMed DOI PMC

Beste DJ V, Bonde B, Hawkins N, Ward JL, Beale MH, Noack S, et al. 13C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation. PLoS Pathog. 2011;7:e1002091 10.1371/journal.ppat.1002091 PubMed DOI PMC

Watanabe S, Zimmermann M, Goodwin MB, Sauer U, Barry CE, Boshoff HI. Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis . PLoS Pathog. 2011;7:e1002287 10.1371/journal.ppat.1002287 PubMed DOI PMC

Marrero J, Rhee KY, Schnappinger D, Pethe K, Ehrt S. Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection. Proc Natl Acad Sci U S A. 2010;107:9819–24. 10.1073/pnas.1000715107 PubMed DOI PMC

Schuck P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J. Elsevier; 2000;78:1606–19. 10.1016/S0006-3495(00)76713-0 PubMed DOI PMC

Schuck P. On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem. 2003;320:104–124. 10.1016/S0003-2697(03)00289-6 PubMed DOI

Kabsch W. XDS. Acta Crystallogr D Biol Crystallogr. 2010;66:125–32. 10.1107/S0907444909047337 PubMed DOI PMC

Krug M, Weiss MS, Heinemann U, Mueller U. XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS. J Appl Crystallogr. 2012;45:568–572. 10.1107/S0021889812011715 DOI

Brünger AT. X-PLOR: version 3.1: a system for x-ray crystallography and NMR Yale Univ Press; 1992;

Lovell SC, Davis IW, Arendall WB, de Bakker PIW, Word JM, Prisant MG, et al. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins. 2003;50:437–50. 10.1002/prot.10286 PubMed DOI

Case DA, Darden TA, Cheatham TE, Simmerling CL, Wang J, Duke RE, et al. AMBER 10. University of California, San Francisco; 2008.

Dapprich S, Komaromi I, Byun KS, Morokuma K, Frisch MJ. A new ONIOM implementation in Gaussian98. Part I. The calculation of energies, gradients, vibrational frequencies and electric field derivatives. J Mol Struct Teochem. 1999;461–462:1–21.

Ahlrichs R, Bär M, Häser M, Horn H, Kölmel C. Electronic structure calculations on workstation computers: The program system turbomole. Chem Phys Lett. 1989;162:165–169. 10.1016/0009-2614(89)85118-8 DOI

Stewart JJP. MOPAC2009. Colorado Springs, CO, USA: Stewart Computational Chemistry;

Jurecka P, Cerný J, Hobza P, Salahub DR. Density functional theory augmented with an empirical dispersion term. Interaction energies and geometries of 80 noncovalent complexes compared with ab initio quantum mechanics calculations. J Comput Chem. 2007;28:555–69. 10.1002/jcc.20570 PubMed DOI

Grimme S, Antony J, Ehrlich S, Krieg H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J Chem Phys. 2010;132:154104 10.1063/1.3382344 PubMed DOI

Řezáč J, Hobza P. Advanced Corrections of Hydrogen Bonding and Dispersion for Semiempirical Quantum Mechanical Methods. J Chem Theory Comput. 2012;8:141–151. 10.1021/ct200751e PubMed DOI

Řezáč J, Fanfrlík J, Salahub D, Hobza P. Semiempirical Quantum Chemical PM6 Method Augmented by Dispersion and H-Bonding Correction Terms Reliably Describes Various Types of Noncovalent Complexes. J Chem Theory Comput. 2009;5:1749–1760. 10.1021/ct9000922 PubMed DOI

Klamt A, Schuurmann G. COSMO: a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J Chem Soc Perkin Trans 2. 1993;799 10.1039/p29930000799 DOI

Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of a general amber force field. J Comput Chem. 2004;25:1157–74. 10.1002/jcc.20035 PubMed DOI

Bradbrook GM, Gleichmann T, Harrop SJ, Habash J, Raftery J, Kalb (Gilboa) J, et al. X-Ray and molecular dynamics studies of concanavalin-A glucoside and mannoside complexes Relating structure to thermodynamics of binding. J Chem Soc Faraday Trans. 1998;94:1603–1611. 10.1039/a800429c DOI

Bayly CI, Cieplak P, Cornell W, Kollman PA. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J Phys Chem. 1993;97:10269–10280. 10.1021/j100142a004 DOI

Massova I, Kollman PA. Computational Alanine Scanning To Probe Protein−Protein Interactions: A Novel Approach To Evaluate Binding Free Energies. J Am Chem Soc. 1999;121:8133–8143. 10.1021/ja990935j DOI

Pecina A, Lepšík M, Řezáč J, Brynda J, Mader P, Řezáčová P, et al. QM/MM calculations reveal the different nature of the interaction of two carborane-based sulfamide inhibitors of human carbonic anhydrase II. J Phys Chem B. 2013;117:16096–104. 10.1021/jp410216m PubMed DOI

Lobley A, Whitmore L, Wallace BA. Bioinformatics applications note. 2002;18:211–212. PubMed

Holten DD, Nordlie RC. Comparative studies of catalytic properties of guinea pig liver intra- and extramitochondrial phosphoenolpyruvate carboxykinases. Biochemistry. 1965;4:723–31. PubMed

Foster DO, Lardy HA, Ray PD, Johnston JB. Alteration of rat liver phosphoenolpyruvate carboxykinase activity by L-tryptophan in vivo and metals in vitro. Biochemistry. 1967;6:2120–8. PubMed

Utter MF, Kolenbrander HM, Boyer ED ed. The Enzymes. Acad Press; New York: 1972;6:136–154.

Matte A, Goldie H, Sweet RM, Delbaere LT. Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold. J Mol Biol. 1996;256:126–43. 10.1006/jmbi.1996.0072 PubMed DOI

Sudom AM, Prasad L, Goldie H, Delbaere LT. The phosphoryl-transfer mechanism of Escherichia coli phosphoenolpyruvate carboxykinase from the use of AlF(3). J Mol Biol. 2001;314:83–92. 10.1006/jmbi.2001.5120 PubMed DOI

Trapani S, Linss J, Goldenberg S, Fischer H, Craievich AF, Oliva G. Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 A resolution. J Mol Biol. 2001;313:1059–72. 10.1006/jmbi.2001.5093 PubMed DOI

Jabalquinto AM, Laivenieks M, González-Nilo FD, Encinas MV, Zeikus G, Cardemil E. Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase: mutagenesis at metal site 2. J Protein Chem. 2003;22:515–9. PubMed

Shi L, Sohaskey CD, Pfeiffer C, Datta P, Parks M, McFadden J, et al. Carbon flux rerouting during Mycobacterium tuberculosis growth arrest. Mol Microbiol. 2010;78:1199–215. 10.1111/j.1365-2958.2010.07399.x PubMed DOI PMC

Urbina JA. The phosphoenolpyruvate carboxykinase of Trypanosoma (Schizotrypanum) cruzi epimastigotes: Molecular, kinetic, and regulatory properties. Arch Biochem Biophys. 1987;258:186–195. 10.1016/0003-9861(87)90335-3 PubMed DOI

Finnegan PM, N. BJ. Isolation and sequence analysis of cDNAs encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides . Plant Mol Biol. Springer; 27:365–376. PubMed

Gobin J, Horwitz MA. Exochelins of Mycobacterium tuberculosis remove iron from human iron-binding proteins and donate iron to mycobactins in the M. tuberculosis cell wall. J Exp Med. 1996;183:1527–32. PubMed PMC

Wagner D, Maser J, Lai B, Cai Z, Barry CE, Höner Zu Bentrup K, et al. Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell’s endosomal system. J Immunol. 2005;174:1491–500. PubMed

Bentle L, Lardy H. Interaction of anions and divalent metal ions with phosphoenolpyruvate carboxykinase. J Biol Chem. 1976;251:2916–21. PubMed

Bentley LA, Lardy A. P-enolpyruvate Ferroactivator. JBC. 1977;252:1431–1440. PubMed

MacDonald MJ. Rapid inactivation of rat liver phosphoenolpyruvate carboxykinase by microsomes and reversal by reductants. Biochim Biophys Acta—Enzymol. 1980;615:223–236. 10.1016/0005-2744(80)90025-X PubMed DOI

MacDonald M, Huang M, Lardy H. Hyperglycaemic activity and metabolic effects of 3-aminopicolinic acid. Biochem J. 1978;495–504. PubMed PMC

MacDonald MJ, Bentle LA, Lardy HA. P-enolpyruvate carboxykinase ferroactivator. Distribution, and the influence of diabetes and starvation. J Biol Chem. 1978;253:116–24. PubMed

MacDonald MJ, Lardy HA. 3-Aminopicolinate: A Synthetic Metal-complexing of Phosphoenolpyruvate Carboxykinase Activator. JBC. 1978;253:2300–2307. PubMed

Punekars NS, Lardy HA. Phosphoenolpyruvate Carboxykinase Ferroactivator 1—I. JBC. 1987;262:6714–6719. PubMed

Via LE, Lin PL, Ray SM, Carrillo J, Allen SS, Eum SY, et al. Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates. Infect Immun. 2008;76:2333–40. 10.1128/IAI.01515-07 PubMed DOI PMC

Abramovitch RB, Rohde KH, Hsu F-F, Russell DG. aprABC: a Mycobacterium tuberculosis complex-specific locus that modulates pH-driven adaptation to the macrophage phagosome. Mol Microbiol. 2011;80:678–94. 10.1111/j.1365-2958.2011.07601.x PubMed DOI PMC

Sherman DR, Voskuil M, Schnappinger D, Liao R, Harrell MI, Schoolnik GK. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha-crystallin. Proc Natl Acad Sci U S A. 2001;98:7534–9. 10.1073/pnas.121172498 PubMed DOI PMC

Sullivan SM, Holyoak T. Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection. Proc Natl Acad Sci U S A. 2008;105:13829–34. 10.1073/pnas.0805364105 PubMed DOI PMC

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