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

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 CDPcholine 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 tRNA 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 Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme. Structured digital abstract• PfCCT MDK and PfCCT MDK bind by mass spectrometry studies of complexes (View interaction)• PfCCT MDK and PfCCT MDK bind by comigration in gel electrophoresis (View interaction)• PfCCT MDK and PfCCT MDK bind by molecular sieving (View interaction)

CTP:phosphocholine cytidylyltransferase: Function, regulation, and structure of an amphitropic enzyme required for membrane biogenesis

“…CCTs from several Plasmodium species are monomers with two full repeats of the four regions (N/C/M/P) shown in Fig. 2, instead of the homodimer arrangement common to most CCTs [224]. The dimerization interface and tertiary structure are likely very similar to the homodimeric CCTs, as shown by recent modeling and MD simulations of the P. falciparum CCT [224,225].…”

“…2, instead of the homodimer arrangement common to most CCTs [224]. The dimerization interface and tertiary structure are likely very similar to the homodimeric CCTs, as shown by recent modeling and MD simulations of the P. falciparum CCT [224,225]. The CCT catalytic domain is highly conserved, and the structure of the rat catalytic domain is a very good model for other CCT catalytic domains [224].…”

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase

“…The R681H mutant construct of His-tagged Pf CCT MΔK (528–795, Δ720–737) was produced by site-directed mutagenesis [10] (PlasmoDB accession number: PF3D7_1316600) [51] using the QuikChange method (Agilent) (for more details about sequence of the protein used for in vitro studies see Fig. A in S2 File).…”

Accurate modeling of cation–π interactions in enzymes: a case study on the CDPCho:phosphocholine cytidylyltransferase complex

“…[26,27] 2.2 MD simulation in enzymes. No crystal structure of the PfCCT enzyme exists, therefore a previously published homology model (PMDB: PM0078719) [28] of the native enzyme product(CDPCho) complex was used as a starting structure for our MD simulations. In the case of the point-mutated enzymes, the Trp692 residue was mutated in silico to Tyr and Phe residues.…”

“…Topology file and parameters for the CDPCho ligand were taken from our previous publication. [28] It should be noted that the parameters for the choline group of the ligand are identical to those from the CHARMM lipid force field. …”