Extended data: Structural and Functional Impact of the G340S Mutation in Plasmodium falciparum Ferredoxin NADP⁺ Reductase: In silico Analysis and Molecular Docking.

Plasmodium falciparum, the deadliest malaria parasite, continues to burden health systems across sub-Saharan Africa. To reduce this burden, Artemisinin-based combination therapies (ACTs), particularly artemether-lumefantrine (AL), remain the frontline treatment. However, partial artemisinin resistance is spreading, exerting selection pressure on lumefantrine (LM), an essential long-acting partner drug. While LM has been described as refractory to resistance, its efficacy could be compromised if resistance arises. To elucidate resistance markers associated with LM, our preliminary studies mapped a mutation in Plasmodium berghei Ferredoxin NADP⁺ Reductase (FNR), G332S, corresponding to G340S in P. falciparum. PfFNR is an essential apicoplast enzyme central to key biosynthetic pathways and drug activation. To explore the structural and functional impact of the G340S substitution, this study employed an in silico bioinformatics approach. High-confidence wild-type and mutant PfFNR models were built using AlphaFold3 and I-TASSER, docking was conducted using AutoDock Vina and AlphaFold3, and molecular dynamics (MD) simulations in GROMACS. Structural visualization and interaction mapping were performed in ChimeraX.  Structural analysis indicates that G340S changes the conformation of the NADP⁺-binding loop, disrupts hydrogen-bonding networks in the cofactor-overlapping site, and decreases the cofactor-binding affinity without perturbing the overall fold. Docking revealed conserved NADP⁺ and ferredoxin binding with localized disturbances that could influence electron transfer. MD simulations revealed a differential flexibility and stability of the mutant compared to the wild-type PfFNR. Single-residue substitution within critical FNR motifs affects NADPH binding enzyme function; thus, G340S may impact enzyme catalysis, alter parasite fitness, and drug susceptibility. Experimental validations are required to confirm these predictions and assess PfFNR potential as both a biomarker and drug target in malaria control.