title: Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active.
abstract: The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis-Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.
authors.affiliation: Laboratory Dynamique des Interactions Membranaires Normales et Pathologiques Université Montpellier 2, Centre National de la Recherche Scientifique, UMR 5235, Place Eugène Bataillon, 34095 Montpellier, France.
authors.forename: Sweta
authors.initials: S
authors.lastname: Maheshwari
authors.forename: Marina
authors.initials: M
authors.lastname: Lavigne
authors.forename: Alicia
authors.initials: A
authors.lastname: Contet
authors.forename: Blandine
authors.initials: B
authors.lastname: Alberge
authors.forename: Emilie
authors.initials: E
authors.lastname: Pihan
authors.forename: Clemens
authors.initials: C
authors.lastname: Kocken
authors.forename: Kai
authors.initials: K
authors.lastname: Wengelnik
authors.forename: Dominique
authors.initials: D
authors.lastname: Douguet
authors.forename: Henri
authors.initials: H
authors.lastname: Vial
authors.forename: Rachel
authors.initials: R
authors.lastname: Cerdan
mesh_terms: D000818:Animals; D001665:Binding Sites; D005260:Female; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D010714:Phosphatidylethanolamines; D015800:Protozoan Proteins; C483858:phosphatidylethanolamine; D012316:RNA Nucleotidyltransferases; C020126:Ethanolamine-phosphate cytidylyltransferase