title: The human malaria parasite Plasmodium falciparum expresses an atypical N-terminally extended pyrophosphokinase with specificity for thiamine.
abstract: Vitamin B(1) is an essential cofactor for key enzymes such as 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. Plants, bacteria and fungi, as well as Plasmodium falciparum, are capable of synthesising vitamin B(1)de novo, whereas mammals have to take up this cofactor from their diet. Thiamine, a B(1) vitamer, has to be pyrophosphorylated by thiamine pyrophosphokinase (TPK) to the active form. The human malaria parasite P. falciparum expresses an N-terminally extended pyrophosphokinase throughout the entire erythrocytic life cycle, which was analysed by Northern and Western blotting. The recombinant enzyme shows a specific activity of 27 nmol min(-1) mg(-1) protein and specificity for thiamine with a K(m) value of 73 microM, while thiamine monophosphate is not accepted. Mutational analysis of the N-terminal extension of the plasmodial TPK showed that it influences thiamine binding as well as metal dependence, which suggests N-terminal participation in the conformation of the active site. Protein sequences of various plasmodial TPKs were analysed for their phylogeny, which classified the Plasmodium TPKs to a group distinct from the mammalian TPKs. To verify the location of the parasite TPK within the cell, immunofluorescence analyses were performed. Co-staining of PfTPK with a GFP marker visualised its cytosolic localisation.
authors.affiliation: Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany.
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Eschbach
authors.forename: Ingrid B
authors.initials: IB
authors.lastname: Müller
authors.forename: Tim-Wolf
authors.initials: TW
authors.lastname: Gilberger
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Walter
authors.forename: Carsten
authors.initials: C
authors.lastname: Wrenger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013836:Thiamin Pyrophosphokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013831:Thiamine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D013836:Thiamin Pyrophosphokinase; D013831:Thiamine