Structural determinants of the catalytic mechanism of Plasmodium CCT, a key enzyme of malaria lipid biosynthesis

Abstract

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.

Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme

“…An 18 amino acid-long, lysine-rich insertion is present in the catalytically important L5 loop in both the first and second catalytic domains (C1 and C2) of Pf CCT, respectively 11 . The L5 loop is responsible for the coordination of the quaternary ammonium moiety of choline with residues Y131/Y714, N133/N716 and Y158/Y741 in C1/C2 and contributes to the formation of the composite aromatic box cleft 43 , 44 . We generated a homology model of Pf CCT to complete the recently resolved crystal structure (PDB: 4ZCS, Fig.…”

“…We generated a homology model of Pf CCT to complete the recently resolved crystal structure (PDB: 4ZCS, Fig. 2 B) 44 by visualizing the 720–737 Lys-rich insertion that was deleted from the crystallized construct. The insertion appears as a highly flexible region, characterized by low QMEAN local quality scores with an average of 0.4.…”

“…The duplication of catalytic and membrane-binding domains as well as the presence of Plasmodium- specific segments distinguishes Pf CCT from its metazoan CCT homologs and the functional relevance of its many traits are yet unexplored. While structural and biochemical studies delineated the catalytic mechanism of Pf CCT 12 , 43 , 44 , the precise regulatory mechanism including membrane binding and compartmentalization for this enzyme is less understood. Here, we used a mammalian cellular system based on the thermosensitive CCT-mutant CHO-MT58 to evaluate the function of the Plasmodium- specific structural elements of the Pf CCT protein.…”

Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface

“…7B). Guca et al (19) had found evidence for phosphocholine-independent PP i production by a catalytically compromised K122A analog in the CCT from P. falciparum, suggesting that CTP hydrolysis might in some cases be a competing reaction for the cytidyl transfer reaction. A hydrolysis potential has never been established for CCT, but it could explain why, for example, the soluble form and CCT-236 have such high apparent K m values for CTP.…”

“…An alternative hypothesis for enhanced catalytic function is that a relatively anhydrous active site would strengthen the charge-charge interactions between enzyme and substrates. The CCT active site provides multiple amine-containing residues that overcome the repulsive negative charges between the substrates, CTP and phosphocholine (19,20) (Fig. 7A).…”