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Evolutionary and mechanistic insights into substrate and product accommodation of CTP :phosphocholine cytidylyltransferase from P lasmodium falciparum
Author(s) -
Nagy Gergely N.,
Marton Lívia,
Krámos Balázs,
Oláh Julianna,
Révész Ágnes,
Vékey Károly,
Delsuc Frédéric,
HunyadiGulyás Éva,
Medzihradszky Katalin F.,
Lavigne Marina,
Vial Henri,
Cerdan Rachel,
Vértessy Beáta G.
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12282
Subject(s) - phosphocholine , isothermal titration calorimetry , chemistry , enzyme , biochemistry , stereochemistry , active site , cofactor , substrate analog , phospholipid , membrane , phosphatidylcholine
The enzyme CTP:phosphocholine cytidylyltransferase ( CCT ) is essential in the lipid biosynthesis of P lasmodia ( H aemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of P lasmodium 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 Mg 2+ , 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 :Mg 2+ , similarly to the related enzyme family of aminoacyl t RNA synthetases, suggests that, with lack of Mg 2+ , 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 P lasmodium 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 ( Pf CCT(528–795)) and PM0078719 ( Pf CCT MΔK) Structured digital abstractPfCCT 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 )