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

Gergely N. Nagy, Lívia Marton, Balázs Krámos, J. Oláh, Ágnes Révész, K. Vékey, Frédéric Delsuc, E. Hunyadi-Gulyás, Katalin F. Medzihradszky, Marina Lavigne, Henri Vial, Rachel Cerdan, B. Vértessy

Research output: Contribution to journalArticle

13 Citations (Scopus)

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. Database Model data are available in the Protein Model DataBase (PMDB) under the accession number PM0078718 (PfCCT(528-795)) and PM0078719 (PfCCT MΔK) Structured digital abstract PfCCT MΔK and PfCCT MΔK bind by mass spectrometry studies of complexes (View interaction) PfCCT MΔK and PfCCT MΔK bind by comigration in gel electrophoresis (View interaction) PfCCT MΔK and PfCCT MΔK bind by molecular sieving (View interaction) CTP:phosphocholine cytidylyltransferase (CCT) is essential in lipid biosynthesis of Plasmodia, presenting a promising antimalarial target. We identified two independent gene duplication events of CCT within Apicomplexa and characterized a dimeric truncated construct of Plasmodium falciparum CCT. Catalytic mechanism of PfCCT may involve conformational changes affecting the choline subsite of the enzyme whereas Mg2+ cofactor is dispensable for CTP substrate binding.

Original languageEnglish
Pages (from-to)3132-3148
Number of pages17
JournalFEBS Journal
Volume280
Issue number13
DOIs
Publication statusPublished - Jul 2013

Fingerprint

Choline-Phosphate Cytidylyltransferase
Plasmodium falciparum
Cytidine Triphosphate
Substrates
Enzymes
Cytidine Diphosphate Choline
Apicomplexa
Gene Duplication
Phosphorylcholine
Plasmodium
Biosynthesis
Antimalarials
Choline
Haemosporida
Catalytic Domain
Genes
Lipids
Amino Acyl-tRNA Synthetases
Enzyme kinetics
Protein Databases

Keywords

  • CTP:phosphocholine cytidylyltransferase
  • gene duplication
  • lipid biosynthesis
  • malaria
  • thermodynamics of ligand binding

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Evolutionary and mechanistic insights into substrate and product accommodation of CTP : phosphocholine cytidylyltransferase from Plasmodium falciparum. / Nagy, Gergely N.; Marton, Lívia; Krámos, Balázs; Oláh, J.; Révész, Ágnes; Vékey, K.; Delsuc, Frédéric; Hunyadi-Gulyás, E.; Medzihradszky, Katalin F.; Lavigne, Marina; Vial, Henri; Cerdan, Rachel; Vértessy, B.

In: FEBS Journal, Vol. 280, No. 13, 07.2013, p. 3132-3148.

Research output: Contribution to journalArticle

Nagy, Gergely N. ; Marton, Lívia ; Krámos, Balázs ; Oláh, J. ; Révész, Ágnes ; Vékey, K. ; Delsuc, Frédéric ; Hunyadi-Gulyás, E. ; Medzihradszky, Katalin F. ; Lavigne, Marina ; Vial, Henri ; Cerdan, Rachel ; Vértessy, B. / Evolutionary and mechanistic insights into substrate and product accommodation of CTP : phosphocholine cytidylyltransferase from Plasmodium falciparum. In: FEBS Journal. 2013 ; Vol. 280, No. 13. pp. 3132-3148.
@article{04b2ef02fd4a44629dec49eeca2bfe5e,
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. Database Model data are available in the Protein Model DataBase (PMDB) under the accession number PM0078718 (PfCCT(528-795)) and PM0078719 (PfCCT MΔK) Structured digital abstract PfCCT MΔK and PfCCT MΔK bind by mass spectrometry studies of complexes (View interaction) PfCCT MΔK and PfCCT MΔK bind by comigration in gel electrophoresis (View interaction) PfCCT MΔK and PfCCT MΔK bind by molecular sieving (View interaction) CTP:phosphocholine cytidylyltransferase (CCT) is essential in lipid biosynthesis of Plasmodia, presenting a promising antimalarial target. We identified two independent gene duplication events of CCT within Apicomplexa and characterized a dimeric truncated construct of Plasmodium falciparum CCT. Catalytic mechanism of PfCCT may involve conformational changes affecting the choline subsite of the enzyme whereas Mg2+ cofactor is dispensable for CTP substrate binding.",
keywords = "CTP:phosphocholine cytidylyltransferase, gene duplication, lipid biosynthesis, malaria, thermodynamics of ligand binding",
author = "Nagy, {Gergely N.} and L{\'i}via Marton and Bal{\'a}zs Kr{\'a}mos and J. Ol{\'a}h and {\'A}gnes R{\'e}v{\'e}sz and K. V{\'e}key and Fr{\'e}d{\'e}ric Delsuc and E. Hunyadi-Guly{\'a}s and Medzihradszky, {Katalin F.} and Marina Lavigne and Henri Vial and Rachel Cerdan and B. V{\'e}rtessy",
year = "2013",
month = "7",
doi = "10.1111/febs.12282",
language = "English",
volume = "280",
pages = "3132--3148",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "13",

}

TY - JOUR

T1 - Evolutionary and mechanistic insights into substrate and product accommodation of CTP

T2 - phosphocholine cytidylyltransferase from Plasmodium falciparum

AU - Nagy, Gergely N.

AU - Marton, Lívia

AU - Krámos, Balázs

AU - Oláh, J.

AU - Révész, Ágnes

AU - Vékey, K.

AU - Delsuc, Frédéric

AU - Hunyadi-Gulyás, E.

AU - Medzihradszky, Katalin F.

AU - Lavigne, Marina

AU - Vial, Henri

AU - Cerdan, Rachel

AU - Vértessy, B.

PY - 2013/7

Y1 - 2013/7

N2 - 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. Database Model data are available in the Protein Model DataBase (PMDB) under the accession number PM0078718 (PfCCT(528-795)) and PM0078719 (PfCCT MΔK) Structured digital abstract PfCCT MΔK and PfCCT MΔK bind by mass spectrometry studies of complexes (View interaction) PfCCT MΔK and PfCCT MΔK bind by comigration in gel electrophoresis (View interaction) PfCCT MΔK and PfCCT MΔK bind by molecular sieving (View interaction) CTP:phosphocholine cytidylyltransferase (CCT) is essential in lipid biosynthesis of Plasmodia, presenting a promising antimalarial target. We identified two independent gene duplication events of CCT within Apicomplexa and characterized a dimeric truncated construct of Plasmodium falciparum CCT. Catalytic mechanism of PfCCT may involve conformational changes affecting the choline subsite of the enzyme whereas Mg2+ cofactor is dispensable for CTP substrate binding.

AB - 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. Database Model data are available in the Protein Model DataBase (PMDB) under the accession number PM0078718 (PfCCT(528-795)) and PM0078719 (PfCCT MΔK) Structured digital abstract PfCCT MΔK and PfCCT MΔK bind by mass spectrometry studies of complexes (View interaction) PfCCT MΔK and PfCCT MΔK bind by comigration in gel electrophoresis (View interaction) PfCCT MΔK and PfCCT MΔK bind by molecular sieving (View interaction) CTP:phosphocholine cytidylyltransferase (CCT) is essential in lipid biosynthesis of Plasmodia, presenting a promising antimalarial target. We identified two independent gene duplication events of CCT within Apicomplexa and characterized a dimeric truncated construct of Plasmodium falciparum CCT. Catalytic mechanism of PfCCT may involve conformational changes affecting the choline subsite of the enzyme whereas Mg2+ cofactor is dispensable for CTP substrate binding.

KW - CTP:phosphocholine cytidylyltransferase

KW - gene duplication

KW - lipid biosynthesis

KW - malaria

KW - thermodynamics of ligand binding

UR - http://www.scopus.com/inward/record.url?scp=84879208183&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84879208183&partnerID=8YFLogxK

U2 - 10.1111/febs.12282

DO - 10.1111/febs.12282

M3 - Article

C2 - 23578277

AN - SCOPUS:84879208183

VL - 280

SP - 3132

EP - 3148

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 13

ER -