title: 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 CDP-choline product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of 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, with 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 indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary. gnagy@enzim.hu
authors.forename: Gergely N
authors.initials: GN
authors.lastname: Nagy
authors.forename: Lívia
authors.initials: L
authors.lastname: Marton
authors.forename: Balázs
authors.initials: B
authors.lastname: Krámos
authors.forename: Julianna
authors.initials: J
authors.lastname: Oláh
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Révész
authors.forename: Károly
authors.initials: K
authors.lastname: Vékey
authors.forename: Frédéric
authors.initials: F
authors.lastname: Delsuc
authors.forename: Éva
authors.initials: É
authors.lastname: Hunyadi-Gulyás
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: Marina
authors.initials: M
authors.lastname: Lavigne
authors.forename: Henri
authors.initials: H
authors.lastname: Vial
authors.forename: Rachel
authors.initials: R
authors.lastname: Cerdan
authors.forename: Beáta G
authors.initials: BG
authors.lastname: Vértessy
mesh_terms: D000595:Amino Acid Sequence; D016782:Apicomplexa / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003566:Cytidine Diphosphate Choline / Q000737:chemistry / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D019143:Evolution, Molecular; D017353:Gene Deletion; D020440:Gene Duplication; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010767:Phosphorylcholine / Q000737:chemistry / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry
chemical_list: D015800:Protozoan Proteins; D010767:Phosphorylcholine; D003566:Cytidine Diphosphate Choline; D003570:Cytidine Triphosphate; D014364:Tryptophan; D019916:Choline-Phosphate Cytidylyltransferase; D008274:Magnesium