In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-D D-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP + reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP + reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.

1 SUMMARYThe completion of the Plasmodium falciparum genome sequence has recently promoted the search for new antimalarial drugs. More specifically, metabolic pathways of the apicoplast, a key organelle for survival of the parasite, have been recognized as potential targets for the development of specific new antimalarial agents. As most apicomplexan parasites, P. falciparum displays a plant-type ferredoxin-NADP + reductase, yielding reduced ferredoxin for essential biosynthetic pathways in the apicoplast. Here we report a molecular, kinetic and ligand binding characterization of the recombinant ferredoxin-NADP + reductase from P. falciparum, at the light of current data available for plant ferredoxin-NADP + reductases. In parallel with the functional characterization, we describe the crystal structures of P. falciparum ferredoxin-NADP + reductase in free form and in complex with 2'-phospho-AMP (at 2.4 and 2.7 Å resolution, respectively).The enzyme displays structural properties likely to be unique of plasmodial reductases. In particular, the two crystal structures highlight a covalent dimer, which relies on the oxidation of residue Cys99 in two opposing subunits, and a helix-coil transition occurs in the NADP-binding domain triggered by 2'-phospho-AMP binding. Studies in solution show that NADP + , as well as 2'-phospho-AMP, promotes the formation of the disulfide-stabilized dimer. The isolated dimer is essentially inactive, but full activity is recovered upon disulfide reduction. The occurrence of residues unique to the plasmodial enzyme, and the discovery of specific conformational properties, highlights the NADP-binding domain of P. falciparum ferredoxin-NADP + reductase as particularly suited for the rational development of antimalarial compounds.2

In the apicoplast of apicomplexan parasites, plastidic-type ferredoxin and ferredoxin-NADP + reductase (FNR) form a short electron transport chain that provides reducing power for the synthesis of isoprenoid precursors. These proteins are attractive targets for the development of novel drugs against diseases such as malaria, toxoplasmosis, and coccidiosis. We have obtained ferredoxin and FNR of both Toxoplasma gondii and Plasmodium falciparum in recombinant form, and recently we solved the crystal structure of the P. falciparum reductase. Here we report on the functional properties of the latter enzyme, which differ markedly from those of homologous FNRs. In the physiological reaction, P. falciparum FNR displays a k cat five-fold lower than those usually determined for plastidic-type FNRs. By rapid kinetics, we found that hydride transfer between NADPH and proteinbound FAD is slower in the P. falciparum enzyme. The redox properties of the enzyme were determined, and showed that the FAD semiquinone species is highly destabilized. We propose that these two features, i.e. slow hydride transfer and unstable FAD semiquinone, are responsible for the poor catalytic efficiency of the P. falciparum enzyme. Another unprecedented feature of the malarial parasite FNR is its ability to yield, under oxidizing conditions, an inactive dimeric form stabilized by an intermolecular disulfide bond. Here we show that the monomer-dimer interconversion can be controlled by oxidizing and reducing agents that are possibly present within the apicoplast, such as H 2 O 2 , glutathione, and lipoate. This finding suggests that modulation of the quaternary structure of P. falciparum FNR might represent a regulatory mechanism, although this needs to be verified in vivo.