Plasmodium falciparum ferredoxin-NADP+ reductase : a plant-type enzyme as a promising new target for novel antimalarial drugs

INTRODUCTION: Plant-type ferredoxin-NADP+ reductases (FNRs) are a widespread group of flavin-dependent enzymes that catalyze the exchange of reducing equivalents between NADP(H) and ferredoxin (Fd) (1). FNRs have been found in plant plastids, cyanobacteria and some eubacteria (1), where they form electron transport chains involved in biosynthetic processes as diverse as photosynthesis, nitrogen assimilation, and response against reactive oxygen species. FNRs have been recently identified in the apicoplast of apicomplexan parasites (2, 3), which includes the causative agents of malaria and toxoplasmosis. Plasmodium falciparum FNR (PfFNR) and Fd (PfFd) have been cloned and functionally characterized (4-6). The PfFNR/PfFd couple has been shown to support in vitro the activity of LytB (6), the last enzyme of the biosynthetic pathway for isoprenoid precursors, which is the site of action of known antiplasmodial compounds. On this basis, PfFNR has been proposed as a possible new target for antimalarial drugs (3). RESULTS: The structure of PfFNR has been determined by X-ray crystallography. Compared to other plastidic-type FNRs, PfFNR displays a significantly lower catalytic efficiency and lower selectivity against NADH. These functional features are probably the consequence of the lack of protein positively-charged groups stabilizing the 2’-phosphate of bound substrate. PfFNR interacts with the adenine moiety of the bound NADP(H) through a His residues not conserved in other FNRs. The role of this residue in catalysis has been investigated by site-directed mutagenesis. NADP(H)-binding to PfFNR occurs through an induced-fit mechanism unprecedented in other enzyme of this protein family. The conformational changes induced by binding to the enzyme of 2’-P-AMP, a NADP analogue, include the partial disruption of an α-helix localized in the NADP-binding domain. Furthermore, PfFNR was shown to undergo NADP+-triggered homodimerization in vitro, resulting in the formation of an intermolecular disulfide bridge and leading to enzyme inactivation. This process, which can be fully reversed by cleaving the disulfide by reductants like DTT or lipoate, could represent a physiologic mechanism regulating the enzyme activity. Structure-based design of PfFNR inhibitors is in progress and has already yielded some active compounds. 1. Ceccarelli E.A. et al. (2004) Biochim. Biophys. Acta 1698, 155-165 2. Pandini V. et al. (2002) J. Biol. Chem. 277, 48463-48471 3. Seeber F. et al. (2005) Curr. Farm. Des. 11, 3159-7312 4. Milani M. et al. (2007) J. Mol. Biol. 367, 501-513 5. Kimata–Ariga Y. et al. (2007) J. Biochem. 142, 715-720 6. Röhrich R.C. et al. (2005) FEBS Lett. 579, 6433–643