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.