The heteromeric Plasmodium falciparum pantothenate kinase has only one active site and does not require Pf14-3-3I 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 PfPanK1, PfPanK2 and Pf14-3-3I. Using a mutagenesis approach, we generated 10 PfPanK mutants and demonstrate that the PfPanK complex has only one functional active site, with both PfPanK1 and PfPanK2 required for activity by the complex. We also show that PfPanK2 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 PfPanK samples revealed phosphorylation sites in both PfPanK1 and PfPanK2 that were additional to the previously reported sites. To investigate the role of specific sites in PfPanK1 and PfPanK2 that may be involved in Pf14-3-3I binding, five additional mutants were generated. Mutagenesis of four predicted Pf14-3-3I binding sites in PfPanK1 resulted in a significant reduction in the amount of Pf14-3-3I bound to the PfPanK complex, with S334 being the most likely binding site. Heterologous expression of the PfPanK 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 Pf14-3-3I may not be essential for PfPanK activity but may be important for stabilising the PfPanK complex.