Comparison of the cellular and biochemical properties of Plasmodium falciparum choline and ethanolamine kinases.
The proliferation of the malaria-causing parasite Plasmodium falciparum within the erythrocyte is concomitant with massive phosphatidylcholine and phosphatidylethanolamine biosynthesis. Based on pharmacological and genetic data, de novo biosynthesis pathways of both phospholipids appear to be essential for parasite survival. The present study characterizes PfCK (P. falciparum choline kinase) and PfEK (P. falciparum ethanolamine kinase), which catalyse the first enzymatic steps of these essential metabolic pathways. Recombinant PfCK and PfEK were expressed as His6-tagged fusion proteins from overexpressing Escherichia coli strains, then purified to homogeneity and characterized. Using murine polyclonal antibodies against recombinant kinases, PfCK and PfEK were shown to be localized within the parasite cytoplasm. Protein expression levels increased during erythrocytic development. PfCK and PfEK appeared to be specific to their respective substrates and followed Michaelis-Menten kinetics. The Km value of PfCK for choline was 135.3+/-15.5 microM. PfCK was also able to phosphorylate ethanolamine with a very low affinity. PfEK was found to be an ethanolamine-specific kinase (Km=475.7+/-80.2 microM for ethanolamine). The quaternary ammonium compound hemicholinium-3 and an ethanolamine analogue, 2-amino-1-butanol, selectively inhibited PfCK or PfEK. In contrast, the bis-thiazolium compound T3, which was designed as a choline analogue and is currently in clinical trials for antimalarial treatment, affected PfCK and PfEK activities similarly. Inhibition exerted by T3 was competitive for both PfCK and PfEK and correlated with the impairment of cellular phosphatidylcholine biosynthesis. Comparative analyses of sequences and structures for both kinase types gave insights into their specific inhibition profiles and into the dual capacity of T3 to inhibit both PfCK and PfEK.
Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier II, Centre National de la Recherche Scientifique, UMR 5235, Montpellier, F-34095, France.
Blandine
B
Alberge
Leila
L
Gannoun-Zaki
Céline
C
Bascunana
Christophe
C
Tran van Ba
Henri
H
Vial
Rachel
R
Cerdan
D000605:Amino Alcohols / Q000494:pharmacology; D000962:Antimalarials / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002384:Catalysis / Q000187:drug effects; D002794:Choline / Q000378:metabolism; D002797:Choline Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D004983:Ethanolamines / Q000378:metabolism; D006426:Hemicholinium 3 / Q000494:pharmacology; D007700:Kinetics; D008856:Microscopy, Fluorescence; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013844:Thiazoles / Q000494:pharmacology
C499114:1,12-bis(4-methyl-5-(2-hydroxyethyl)thiazol-3-ium-3-yl)dodecane; D000605:Amino Alcohols; D000962:Antimalarials; D004983:Ethanolamines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013844:Thiazoles; D006426:Hemicholinium 3; D017853:Phosphotransferases (Alcohol Group Acceptor); D002797:Choline Kinase; C020125:ethanolamine kinase; C006506:2-amino-1-butanol; D002794:Choline