Phosphopyruvate Carboxylase Identified as a Key Enzyme in Erythrocytic Carbon Metabolism
Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery.
Vyšlo v časopise:
Phosphopyruvate Carboxylase Identified as a Key Enzyme in Erythrocytic Carbon Metabolism. PLoS Pathog 10(1): e32767. doi:10.1371/journal.ppat.1003876
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1003876
Souhrn
Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery.
Zdroje
1. WHO, (2012) World Malaria Report 2012, World Health Organisation, Geneva, Switzerland, 59.
2. HaySI, GuerraCA, GethingPW, PatilAP, TatemAJ, et al. (2009) A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med 6: e1000048.
3. MacRaeJI, DixonMW, DearnleyMK, ChuaHH, ChambersJM, et al. (2013) Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum. BMC Biol 11: 67.
4. FothBJ, StimmlerLM, HandmanE, CrabbBS, HodderAN, et al. (2005) The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast. Mol Microbiol 55: 39–53.
5. PeiY, TarunAS, VaughanAM, HermanRW, SolimanJM, et al. (2010) Plasmodium pyruvate dehydrogenase activity is only essential for the parasite's progression from liver infection to blood infection. Mol Microbiol 75: 957–971.
6. CobboldSA, VaughanAM, LewisIA, PainterHJ, CamargoN, et al. (2013) Kinetic flux profiling elucidates two independent acetyl-CoA biosynthetic pathways in Plasmodium falciparum. J Biol Chem Oct 25 [Epub ahead of print].
7. MacRaeJI, SheinerL, NahidA, TonkinC, StriepenB, et al. (2012) Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of Toxoplasma gondii. Cell Host Microbe 12: 682–692.
8. OlszewskiKL, MatherMW, MorriseyJM, GarciaBA, VaidyaAB, et al. (2010) Branched tricarboxylic acid metabolism in Plasmodium falciparum. Nature 466: 774–778.
9. OlszewskiKL, MatherMW, MorriseyJM, GarciaBA, VaidyaAB, et al. (2013) Retraction: Branched tricarboxylic acid metabolism in Plasmodium falciparum. Nature 497: 652.
10. ShermanIW, TingIP (1966) Carbon dioxide fixation in malaria (Plasmodium lophurae). Nature 212: 1387–1388.
11. ShermanIW, TingIP (1968) Carbon dioxide fixation in malaria. II. Plasmodium knowlesi (monkey malaria). Comp Biochem Physiol 24: 639–642.
12. NagarajanK (1968) Metabolism of Plasmodium berghei. 3. Carbon dioxide fixation and role of pyruvate and dicarboxylic acids. Exp Parasitol 22: 33–42.
13. TragerW, JensenJB (1976) Human malaria parasites in continuous culture. Science 193: 673–675.
14. McDanielHG, SiuPM (1972) Purification and characterization of phosphoenolpyruvate carboxylase from Plasmodium berghei. J Bacteriol 109: 385–390.
15. BlumJJ, GinsburgH (1984) Absence of alpha-ketoglutarate dehydrogenase activity and presence of CO2-fixing activity in Plasmodium falciparum grown in vitro in human erythrocytes. J Protozool 31: 167–169.
16. HaywardRE (2000) Plasmodium falciparum phosphoenolpyruvate carboxykinase is developmentally regulated in gametocytes. Mol Biochem Parasitol 107: 227–240.
17. KrungkraiSR, SuraveratumN, RochanakijS, KrungkraiJ (2001) Characterisation of carbonic anhydrase in Plasmodium falciparum. Int J Parasitol 31: 661–668.
18. IzuiK, MatsumuraH, FurumotoT, KaiY (2004) Phosphoenolpyruvate carboxylase: a new era of structural biology. Annu Rev Plant Biol 55: 69–84.
19. O'LearyB, ParkJ, PlaxtonWC (2011) The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem J 436: 15–34.
20. West-EberhardMJ, SmithJA, WinterK (2011) Plant science. Photosynthesis, reorganized. Science 332: 311–312.
21. MaierAG, BraksJA, WatersAP, CowmanAF (2006) Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination. Mol Biochem Parasitol 150: 118–121.
22. PengY, CaiJ, WangW, SuB (2012) Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family. PLoS One 7: e51159.
23. SvenssonP, BläsingOE, WesthoffP (2003) Evolution of C4 phosphoenolpyruvate carboxylase. Arch Biochem Biophys 414: 180–188.
24. WesthoffP, GowikU (2004) Evolution of c4 phosphoenolpyruvate carboxylase. Genes and proteins: a case study with the genus Flaveria. Ann Bot 93: 13–23.
25. TreeckM, SandersJL, EliasJE, BoothroydJC (2011) The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries. Cell Host Microbe 10: 410–419.
26. JanskiAM, CornellNW (1981) Inhibition by cycloserine of mitochondrial and cytosolic aspartate aminotransferase in isolated rat hepatocytes. Biochem J 194: 1027–1030.
27. GujjarR, El MazouniF, WhiteKL, WhiteJ, CreasonS, et al. (2011) Lead optimization of aryl and aralkyl amine-based triazolopyrimidine inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with antimalarial activity in mice. J Med Chem 54: 3935–3949.
28. VaidyaAB, MatherMW (2009) Mitochondrial evolution and functions in malaria parasites. Annu Rev Microbiol 63: 249–267.
29. EsserL, QuinnB, LiYF, ZhangM, ElberryM, et al. (2004) Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex. J Mol Biol 341: 281–302.
30. BartonV, FisherN, BiaginiGA, WardSA, O'NeillPM (2010) Inhibiting Plasmodium cytochrome bc1: a complex issue. Curr Opin Chem Biol 14: 440–446.
31. RibautC, BerryA, ChevalleyS, ReybierK, MorlaisI, et al. (2008) Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species. Malar J 7: 45.
32. JenkinsCL (1989) Effects of the phosphoenolpyruvate carboxylase inhibitor 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)propenoate on photosynthesis: C(4) selectivity and studies on C(4) photosynthesis. Plant Physiol 89: 1231–1237.
33. GardnerMJ, HallN, FungE, WhiteO, BerrimanM, et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419: 498–511.
34. GibalaMJ, YoungME, TaegtmeyerH (2000) Anaplerosis of the citric acid cycle: role in energy metabolism of heart and skeletal muscle. Acta Physiol Scand 168: 657–665.
35. JitrapakdeeS, Vidal-PuigA, WallaceJC (2006) Anaplerotic roles of pyruvate carboxylase in mammalian tissues. Cell Mol Life Sci 63: 843–854.
36. OlszewskiKL, LlinásM (2011) Central carbon metabolism of Plasmodium parasites. Mol Biochem Parasitol 175: 95–103.
37. García-AllesLF, ErniB (2002) Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes. Eur J Biochem 269: 3226–3236.
38. IzuiK, MatsudaY, KameshitaI, KatsukiH, WoodsAE (1983) Phosphoenolpyruvate carboxylase of Escherichia coli. Inhibition by various analogs and homologs of phosphoenolpyruvate. J Biochem 94: 1789–1795.
39. BulusuV, JayaramanV, BalaramH (2011) Metabolic fate of fumarate, a side product of the purine salvage pathway in the intraerythrocytic stages of Plasmodium falciparum. J Biol Chem 286: 9236–9245.
40. TanakaKR, ValentineWN (1961) Fumarase activity of human leukocytes and erythrocytes. Blood 17: 328–333.
41. SimpsonRJ, BrindleKM, CampbellID (1982) Spin ECHO proton NMR studies of the metabolism of malate and fumarate in human erythrocytes. Dependence on free NAD levels. Biochim Biophys Acta 721: 191–200.
42. TripathiAK, DesaiPV, PradhanA, KhanSI, AveryMA, et al. (2004) An alpha-proteobacterial type malate dehydrogenase may complement LDH function in Plasmodium falciparum. Cloning and biochemical characterization of the enzyme. Eur J Biochem 271: 3488–3502.
43. NozawaA, FujimotoR, MatsuokaH, TsuboiT, TozawaY (2011) Cell-free synthesis, reconstitution, and characterization of a mitochondrial dicarboxylate-tricarboxylate carrier of Plasmodium falciparum. Biochem Biophys Res Commun 414: 612–617.
44. van DoorenGG, StimmlerLM, McFaddenGI (2006) Metabolic maps and functions of the Plasmodium mitochondrion. FEMS Microbiol Rev 30: 596–630.
45. WrengerC, MüllerS (2003) Isocitrate dehydrogenase of Plasmodium falciparum. Eur J Biochem 270: 1775–1783.
46. LambrosC, VanderbergJP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418–420.
47. UmlasJ, FallonJN (1971) New thick-film technique for malaria diagnosis. Use of saponin stromatolytic solution for lysis. Am J Trop Med Hyg 20: 527–529.
48. WuY, SifriCD, LeiHH, SuXZ, WellemsTE (1995) Transfection of Plasmodium falciparum within human red blood cells. Proc Natl Acad Sci USA 92: 973–977.
49. KirkmanLA, SuXZ, WellemsTE (1996) Plasmodium falciparum: isolation of large numbers of parasite clones from infected blood samples. Exp Parasitol 83: 147–149.
50. GüntherS, WallaceL, PatzewitzEM, McMillanPJ, StormJ, et al. (2007) Apicoplast lipoic acid protein ligase B is not essential for Plasmodium falciparum. PLoS Pathog 3: e189.
51. DesjardinsRE, CanfieldCJ, HaynesJD, ChulayJD (1979) Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16: 710–718.
52. De SouzaDP, SaundersEC, McConvilleMJ, LikićVA (2006) Progressive peak clustering in GC-MS metabolomic experiments applied to Leishmania parasites. Bioinformatics 22: 1391–1396.
53. TrangDT, HuyNT, KariuT, TajimaK, KameiK (2004) One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells. Malar J 3: 7.
54. AhnSY, ShinMY, KimYA, YooJA, KwakDH, et al. (2008) Magnetic separation: a highly effective method for synchronization of cultured erythrocytic Plasmodium falciparum. Parasitol Res 102: 1195–1200.
55. TautenhahnR, BöttcherC, NeumannS (2008) Highly sensitive feature detection for high resolution LC/MS. BMC Bioinformatics 9: 504.
56. ScheltemaRA, JankevicsA, JansenRC, SwertzMA, BreitlingR (2011) PeakML/mzMatch: a file format, Java library, R library, and tool-chain for mass spectrometry data analysis. Anal Chem 83: 2786–2793.
57. JankevicsA, MerloME, De VriesM, VonkRJ, TakanoE, et al. (2012) Separating the wheat from the chaff: a prioritisation pipeline for the analysis of metabolomics datasets. Metabolomics 8: 29–36.
58. ChokkathukalamA, JankevicsA, CreekDJ, AchcarF, BarrettMP, et al. (2013) mzMatch-ISO: an R tool for the annotation and relative quantification of isotope-labelled mass spectrometry data. Bioinformatics Oxford England 29: 281–283.
59. ZhangY (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 23 9: 40.
60. RoyA, KucukuralA, ZhangY (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5: 725–738.
61. MatsumuraH, XieY, ShirakataS, InoueT, YoshinagaT, et al. (2002) Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases. Structure 10: 1721–1730.
62. DharmarajanL, KraszewskiJL, MukhopadhyayB, DuntenPW (2011) Structure of an archaeal-type phosphoenolpyruvate carboxylase sensitive to inhibition by aspartate. Proteins 79: 1820–1829.
63. GuexN, PeitschMC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714–2723.
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Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
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