Evolutionary and mechanistic insights into substrate and product accommodation of CTP:phosphocholine cytidylyltransferase from Plasmodium falciparum

Gergely N. Nagy, Lívia Marton, Balázs Krámos, Julianna Oláh, Ágnes Révész, Károly Vékey, Frédéric Delsuc, Éva Hunyadi-Gulyás, Katalin F. Medzihradszky, Marina Lavigne, Henri Vial, Rachel Cerdan, Beáta G. Vértessy

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in the lipid biosynthesis of Plasmodia (Haemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of Plasmodium falciparum CCT that forms a dimer resembling the molecular architecture of CCT enzymes from other sources. Based on biophysical and enzyme kinetics methods, our data show that the CDP-choline product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of Mg2+, considered to be a cofactor of the enzyme, the binding of the CTP substrate is attenuated by a factor of 5. The weaker binding of CTP:Mg2+, similarly to the related enzyme family of aminoacyl tRNA synthetases, suggests that, with lack of Mg2+, positively charged side chain(s) of CCT may contribute to CTP accommodation. Thermodynamic investigations by isothermal titration calorimetry and fluorescent spectroscopy studies indicate that accommodation of the choline phosphate moiety in the CCT active site is different when it appears on its own as one of the substrates or when it is linked to the CDP-choline product. A tryptophan residue within the active site is identified as a useful internal fluorescence sensor of enzyme-ligand binding. Results indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme. Database Model data are available in the Protein Model DataBase (PMDB) under the accession number PM0078718 (PfCCT(528-795)) and PM0078719 (PfCCT MΔK) Structured digital abstract PfCCT MΔK and PfCCT MΔK bind by mass spectrometry studies of complexes (View interaction) PfCCT MΔK and PfCCT MΔK bind by comigration in gel electrophoresis (View interaction) PfCCT MΔK and PfCCT MΔK bind by molecular sieving (View interaction) CTP:phosphocholine cytidylyltransferase (CCT) is essential in lipid biosynthesis of Plasmodia, presenting a promising antimalarial target. We identified two independent gene duplication events of CCT within Apicomplexa and characterized a dimeric truncated construct of Plasmodium falciparum CCT. Catalytic mechanism of PfCCT may involve conformational changes affecting the choline subsite of the enzyme whereas Mg2+ cofactor is dispensable for CTP substrate binding.

Original languageEnglish
Pages (from-to)3132-3148
Number of pages17
JournalFEBS Journal
Volume280
Issue number13
DOIs
Publication statusPublished - Jul 1 2013

Keywords

  • CTP:phosphocholine cytidylyltransferase
  • gene duplication
  • lipid biosynthesis
  • malaria
  • thermodynamics of ligand binding

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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