Phosphoenolpyruvate Carboxylase Identified as a Key Enzyme in Erythrocytic Plasmodium falciparum Carbon Metabolism

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10(pepc)), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10(pepc) had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using C-13-U-D-glucose and C-13-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10(pepc) and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of C-13,N-15-U-glutamine was similar in both parasite lines, although the flux was lower in D10(pepc); it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery. Author Summary The genome of the human malaria parasite Plasmodium falciparum encodes a protein called phosphoenolpyruvate carboxylase (PEPC) absent from the human host. PEPC is known to fix CO2 to generate metabolites used for energy metabolism in plants and bacteria, but its function in malaria parasites remained an enigma. Our study aimed to elucidate the role and importance of PEPC in P. falciparum in its host red blood cell by generating a gene deletion mutant in P. falciparum. This was only achievable in the presence of high concentrations of malate were added to the culture medium. The mutant generated (D10(pepc)) had a severe growth defect, which was rescued partially by malate or fumarate (but not any other downstream metabolites), suggesting that they feed into the same metabolic pathway. Using heavy isotope labelled C-13-U-D-glucose and C-13-bicarbonate we showed that PECP has an important role in intermediary carbon metabolism and is vital for the maintenance of cytosolic and mitochondrial redox balance. Together these findings imply that PEPC may be an exploitable target for future drug discovery.