RESUMO
The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.
Assuntos
Colina-Fosfato Citidililtransferase/química , Lipogênese/genética , Malária Falciparum/genética , Plasmodium falciparum/química , Sequência de Aminoácidos/genética , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Catálise , Domínio Catalítico/genética , Colina-Fosfato Citidililtransferase/genética , Humanos , Cinética , Ligantes , Lipídeos/biossíntese , Lipídeos/química , Lipídeos/genética , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Ligação Proteica , Especificidade por SubstratoRESUMO
The plasmodial CTP:phosphocholine cytidylyltransferase (PfCCT) is a promising antimalarial target, which can be inhibited to exploit the need for increased lipid biosynthesis during the erythrocytic life stage of Plasmodium falciparum. Notable structural and regulatory differences of plasmodial and mammalian CCTs offer the possibility to develop species-specific inhibitors. The aim of this study was to use CHO-MT58 cells expressing a temperature-sensitive mutant CCT for the functional characterization of PfCCT. We show that heterologous expression of wild type PfCCT restores the viability of CHO-MT58 cells at non-permissive (40 °C) temperatures, whereas catalytically perturbed or structurally destabilized PfCCT variants fail to provide rescue. Detailed in vitro characterization indicates that the H630N mutation diminishes the catalytic rate constant of PfCCT. The flow cytometry-based rescue assay provides a quantitative readout of the PfCCT function opening the possibility for the functional analysis of PfCCT and the high throughput screening of antimalarial compounds targeting plasmodial CCT.
