The human malaria parasite Plasmodium falciparum expresses an atypical N-terminally extended pyrophosphokinase with specificity for thiamine

Abstract

AbstractVitamin B1is an essential cofactor for key enzymes such as 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. Plants, bacteria and fungi, as well asPlasmodium falciparum, are capable of synthesising vitamin B1de novo, whereas mammals have to take up this cofactor from their diet. Thiamine, a B1vitamer, has to be pyrophosphorylated by thiamine pyrophosphokinase (TPK) to the active form. The human malaria parasiteP. falciparumexpresses 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-1mg-1protein and specificity for thiamine with aKmvalue of 73 μM, 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 thePlasmodiumTPKs 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 ofPfTPK with a GFP marker visualised its cytosolic localisation.