The heteromeric Plasmodium falciparum pantothenate kinase has only one active site and does not require Pf 14-3-3I for activity

Coenzyme A (CoA) is an essential molecule for the intraerythrocytic stage of Plasmodium falciparum . Pantothenate kinase (PanK) catalyses the first step of the CoA biosynthesis pathway and functions as a homodimer in most organisms investigated thus far. P. falciparum possesses a novel heteromeric PanK complex composed of Pf PanK1, Pf PanK2 and Pf 14-3-3I. Using a mutagenesis approach, we generated 10 Pf PanK mutants and demonstrate that the Pf PanK complex has only one functional active site, with both Pf PanK1 and Pf PanK2 required for activity by the complex. We also show that Pf PanK2 is essential for normal intraerythrocytic parasite proliferation using a conditional knockdown system. 14-3-3 binding motifs generally contain a phosphoserine/threonine residue. Mass spectrometry analyses of phospho-peptide enriched, immunoprecipitated Pf PanK samples revealed phosphorylation sites in both Pf PanK1 and Pf PanK2 that were additional to the previously reported sites. To investigate the role of specific sites in Pf PanK1 and Pf PanK2 that may be involved in Pf 14-3-3I binding, five additional mutants were generated. Mutagenesis of four predicted Pf 14-3-3I binding sites in Pf PanK1 resulted in a significant reduction in the amount of Pf 14-3-3I bound to the Pf PanK complex, with S334 being the most likely binding site. Heterologous expression of the Pf PanK complex in an insect cell system yielded a small amount of soluble protein that assembled in situ into a functional complex. Combined results from heterologous expression and P. falciparum mutagenesis suggest that Pf 14-3-3I may not be essential for Pf PanK activity but may be important for stabilising the Pf PanK complex.