The elusive role played by the side chain of Lys249 of Plasmodium falciparum ferredoxin-NADP+ reductase in NADPH/NADH selectivity

Plasmodium falciparum, the causative agent of tropical malaria, possesses plant-type ferredoxin-NADP+ reductase (PfFNR) and ferredoxin (PfFd), which have been proposed as targets for novel antimalarial drugs (1). The catalytic mechanism of PfFNR is under study in order to provide the bases for the design of effective enzyme inhibitors (2). Although PfFNR is quite specific for NADPH, no structural evidence was obtained for protein positive groups interacting with the 2’-phosphate of this substrate (3). We have found that change of Lys249 to Ala decreased the kcat/Km for NADPH by a factor of 10, without affecting the activity of the enzyme with NADH. In addition, PfFNR-K249A displayed an affinity for NADP+ 8-fold lower than those of the wild-type enzyme. We conclude that the side-chain of Lys249 actually interacts with the 2’-phosphate of NADPH during the catalytic cycle, and that it is part of a region flexible enough to adopt in the crystal form of the protein a conformation where such interaction is severed 1. Seeber, F., et al. (2005) Curr. Pharm. Des. 11, 3159-3172. 2. Crobu, D., et al. (2009) Biochemistry 48, 9525-9533. 3. Milani, M., et al. (2007) J. Mol. Biol. 367, 501-513.