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

Nagy N., Gergely and Marton, Lívia and Krámos, Balázs and Oláh, Julianna and Révész, Ágnes and Vékey, Károly and Delsuc, Frederic and Hunyadi-Gulyás, Éva and Medzihradszky, Katalin F. and Lavigne, Marina and Vial, Henri and Cerdan, Rachel and Vértessy G., Beáta (2013) Evolutionary and mechanistic insights into substrate and product accommodation of CTP:phosphocholine cytidylyltransferase from Plasmodium falciparum. FEBS Journal. ISSN 1742-464X

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Abstract

The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in 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 as compared to either substrate (CTP or choline phosphate). Interestingly, in the presence of the 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 in 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 argue that the catalytic mechanism of PfCCT may involve conformational changes affecting the choline subsite of the enzyme. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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