The deletion of a protein hydroxyl interacting with the 2’-phosphate of NADPH abolishes cosubstrate specificity in Plasmodium falciparum ferredoxin-NADP+ reductase

The flavoenzyme ferredoxin-NADP+ reductase (FNR) of Plasmodium falciparum (PfFNR) (1) is highly similar to plant FNRs (2). It is localized in the apicoplast of the malaria parasite, where it generates reduced ferredoxin at the expenses of NADPH to fuel reductive biosynthetic pathways. Since PfFNR is required to support the production of isoprenoid precursors, it is an attractive target of new antimalarials. We are studying structure-function relationships of this enzyme as a prerequisite for the rational design of inhibitors (1, 3). NADPH-binding to PfFNR involves the sandwiching of the adenine by the side-chains of His286 and Tyr258, which also establish H-bonds with the 5’- and 2’-phosphate of the cosubstrate adenylate, respectively (1). To evaluate the catalytic role of the Tyr258 hydroxyl group, we generated and characterized the PfFNR-Y258F variant. This mutation turned out to highly affect the steady-state kinetic parameters of the NADPH- and NADH-dependent reaction catalyzed by PfFNR, with the consequence that the enzyme specificity for NADPH vs NADH, as measured from the kcat/Km ratio, drops from 70:1 to 1.7:1. Furthermore, the specificity of the mutant is completely abolished at high NAD(P)H concentration, since PfFNR-Y258F displays the same kcat of ~100 s-1 with either cosubstrates. The critical role played by Tyr258 in modulating the specificity of PfFNR has been confirmed by ligand-binding studies and by pre-steady state kinetics. To the best of our knowledge, this represent a unique case in the FNR family where a single protein group is almost the sole responsible of cosubstrate specificity.