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Plasmodium falciparummalaria parasites are early-diverging eukaryotes with many unusual metabolic adaptations. Understanding these adaptations will give insight into parasite evolution and unveil new parasite-specific drug targets. Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis. In yeast and humans, mACP also functions to biochemically couple FASII activity to electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast to most eukaryotes, thePlasmodiummitochondrion lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC followed by broader cellular defects. Biochemical studies reveal thatPlasmodiummACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of both Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals thatPlasmodiumparasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of otherApicomplexanpathogens, includingToxoplasmaandBabesia. This discovery also highlights the ancient, fundamental role of ACP in mitochondrial Fe-S cluster biogenesis and unveils an evolutionary driving force to retain this interaction with ACP independent of its eponymous function in FASII.Significance StatementPlasmodiummalaria parasites are single-celled eukaryotes that evolved unusual metabolic adaptations. Parasites require a mitochondrion for blood-stage viability, but essential functions beyond the electron transport chain are sparsely understood. Unlike yeast and human cells, thePlasmodiummitochondrion lacks fatty acid synthesis enzymes but retains a divergent acyl carrier protein (mACP) incapable of tethering acyl groups. Nevertheless, mACP is essential for parasite viability by binding and stabilizing the core mitochondrial Fe-S cluster biogenesis complex via a divergent molecular interface lacking an acyl-pantetheine group that contrasts with other eukaryotes. This discovery unveils an essential metabolic adaptation inPlasmodiumand other human parasites that decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis and evolved at or near the emergence ofApicomplexanparasitism.

Doxycycline (DOX) is a key antimalarial drug that is thought to kill Plasmodium falciparum parasites by blocking protein translation in the essential apicoplast organelle. Although parasite resistance to DOX has not emerged, clinical use is primarily limited to prophylaxis due to delayed second-cycle parasite death at 1-3 μM, the DOX concentration readily achieved in human plasma at current dosing. Slow antiparasitic activity is thought to be a fundamental limitation of DOX and other antibiotics that target apicoplast maintenance. We report that slightly higher 8-10 μM DOX concentrations kill parasites with first-cycle activity that blocks apicoplast biogenesis. This faster activity is rescued by exogenous isopentenyl pyrophosphate, an essential apicoplast product, confirming an apicoplast-specific target. Exogenous iron rescues first- but not second-cycle activity by 1 or 10 μM DOX, revealing that first-cycle activity involves a metal-dependent mechanism that is distinct from the canonical delayed-death mechanism. These results provide a new paradigm for understanding fundamental antiparasitic mechanisms of DOX and suggest the possibility of repurposing DOX as a faster-acting antimalarial at higher dosing, which remains well tolerated. Multiple mechanisms of DOX activity would be expected to limit parasite resistance.

Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations. DOI: http://dx.doi.org/10.7554/eLife.09143.001, eLife digest Malaria is a devastating infectious disease that is caused by single-celled parasites called Plasmodium that can live inside red blood cells. Several important proteins from these parasites require a small molecule called heme in order to work. The parasites have enzymes that make heme via a series of intermediate steps. However, it remains unclear exactly how important this ‘pathway’ of enzymes is for the parasite, and whether this pathway could be targeted by drugs to treat malaria. Now Sigala et al. have used a range of genetic and biochemical approaches to better understand the production of heme molecules in Plasmodium-infected red blood cells. First, several parasite genes that encode the enzymes used to make heme molecules were deleted. Unexpectedly, these gene deletions did not affect the ability of the infected blood cells to make heme. This result suggested that the parasites do not use their own pathway to produce heme while they are growing in the bloodstream. Sigala et al. then showed that human enzymes involved in making heme, most of which are also found within the infected red blood cells, are still active. These human enzymes provide a parallel pathway that can link up with the final parasite enzyme to generate heme. Further experiments revealed that the activity of the human enzymes could be strongly stimulated by providing the pathway with one of the building blocks used to make heme. This stimulation led to the build-up of an intermediate molecule called PPIX. This intermediate molecule can kill cells when it is exposed to light—a property that is called ‘phototoxicity’. Sigala et al. showed that treating infected red blood cells with a new combination of non-toxic chemicals that emit light can activate PPIX in the bloodstream and can selectively kill the malaria parasites while leaving uninfected cells intact. These findings suggest a new treatment that could be effective against blood-stage malaria. Furthermore, the parasite will be unable to easily mutate to avoid the effects of this treatment because it relies on human proteins that are already made. Future work is now needed to optimize the dosage and the combination of drugs that could provide such a treatment. DOI: http://dx.doi.org/10.7554/eLife.09143.002

A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution., Enzymatic reactions require exquisitely detailed molecular interactions. Here the authors show that geometric complementarity is likely more important than electrostatic charge in contributing to the binding necessary for catalytic reactions.