Cofactor metabolism in malaria parasites

Nearly half of the world's population lives at risk of malaria, a disease cause by protozoan parasites of the genus Plasmodium. Widespread resistance to current antimalarial chemotherapies has necessitated the search for novel drug targets. Enzyme cofactors are small molecule ligands attached to proteins that serve numerous structural and catalytic roles. Because cofactors are generally required to activate their partner protein, they represent key regulatory steps for metabolic pathways that may be points of intervention for new antimalarial drugs. We investigated the metabolic roles of two enzyme cofactors in malaria parasites: biotin and iron-sulfur (FeS) clusters.;Biotin acts as a CO2 carrier in carboxylation and decarboxylation reactions. In Chapter 1, we developed a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography. The final step of the E. coli biotin biosynthetic pathway is the insertion of sulfur into desthiobiotin to form biotin, a reaction catalyzed by biotin synthase (BioB). We supplemented E. coli with Na35SO4 and desthiobiotin, and produced [35S]-biotin with a specific activity of 30.7 Ci/mmol. In order to collect the radiolabel, we expressed the biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC). We purified biotinylated PfBCCP-79 by affinity chromatography, and liberated free biotin using acid hydrolysis. We first tested this reagent through reuptake in E. coli, in order to confirm the quality of our radiolabel, and showed that it is biologically active and specifically labels biotinylated proteins. We then used [35S]-biotin in uptake assays in P. falciparum, however we were unable to detect any biotinylated proteins in the blood stages.;Biotin carboxylases are inactive until they are biotinylated by a dedicated biotin ligase. Malaria parasites are unusual in that they appear to contain two biotin ligases, but only a single biotin-dependent carboxylase, acetyl-CoA carboxylase (ACC). ACC should be required for type II fatty acid synthesis (FASII) in the apicoplast, a pathway critical for liver-stage development. ACC localizes to the apicoplast in blood- and liver-stage malaria parasites, but both biotin ligases function in the cytosol. Furthermore, biotinylated proteins can only be detected in the apicoplast of liver-stage parasites, suggesting that ACC is biotinylated only in the liver stages, although it is expressed in both blood and liver stages. This implies that ACC activity is regulated through stage-specific biotinylation. In an independent approach, the biotinylation domain of ACC was used as a genetic probe for biotinylation activity. This domain was robustly biotinylated when expressed in the cytosol, but was not biotinylated in the apicoplast. Additionally, we found that biotin is not required for blood-stage growth, even in media with limited fatty acid content. These results suggest that ACC is not required for any FASII or fatty acid elongation (ELO) pathways in blood-stage parasites. In liver-stage parasites, biotinylation of ACC is presumably linked to the nutritional status of the host since neither the parasite nor the host can synthesize the cofactor.;FeS clusters are inorganic cofactors of a large family of proteins involved in electron transfer, enzyme catalysis and regulation. There are three pathways for FeS cluster biogenesis in eukaryotes: the ISC system is located in mitochondria, the CIA system is in the cytosol, and plastid-containing organisms have the SUF pathway in this compartment. We expect malaria parasites to contain all three pathways, and furthermore we predict that they are all essential for viability. The presence of the SUF pathway in the apicoplast was recently demonstrated, and in this study we provide the first experimental evidence for an ISC pathway in the mitochondrion and a CIA pathway in the cytosol. We localized the ISC cysteine desulfurase complex (PfIscS and its effector protein PfIsd11) to the mitochondrion in P. falciparum, suggesting that the ISC pathway functions in this organelle. We localized one of the CIA scaffold proteins, PfCfdl, to the cytosol, but unexpectedly, the other candidate scaffold protein, PfNbp35, localizes to the mitochondrion. We therefore expect that PfCfdl is the sole CIA scaffold protein in P. falciparum. Thus, we have provided the first experimental evidence describing the organization of biotin metabolism in malaria parasites, and the first data identifying FeS cluster biogenesis pathways in the mitochondrion and cytosol of P. falciparum..