Fluazinam binds with the ATP synthase subunit FfATPh but not with FfATP5 or FfATPb of Fusarium fujikuroi

BACKGROUND Rice bakanae disease, a devastating disease caused by Fusarium fujikuroi infection, is primarily managed through chemical agents. Fluazinam, an oxidative phosphorylation uncoupler, is registered for controlling rice bakanae disease, yet its precise mechanism of action remains unclear. Our previous studies found that deletion mutants of ATP synthase subunits FfATPh, FfATP5, and FfATPb in Fusarium fujikuroi showed reduced sensitivity to fluazinam, therefore we aimed to investigate whether fluazinam is capable of binding with FfATPh, FfATP5, and FfATPb. RESULTS In this study, the ATP synthase subunits FfATPh, FfATP5, and FfATPb were obtained via prokaryotic expression and nickel‐column affinity purification. Microscale thermophoresis (MST) results showed that fluazinam binds with His9‐MBP‐TEV‐ATPh and the K d was 25.34 ± 11.71 nM. In contrast, its K d values for His9‐MBP‐TEV‐ATP5 and His9‐MBP‐TEV‐ATPb were 1.49 ± 1.32 μM and 1.04 ± 0.75 μM, respectively, both similar to that of the His9‐MBP‐TEV control (2.57 ± 2.02 μM). These findings indicate that fluazinam exhibits specific affinity with FfATPh. Molecular docking analysis showed that fluazinam can form hydrogen bonds with Asp42 and Phe43 of FfATPh. Further affinity measurements of purified FfATPh point‐mutant proteins showed that the binding affinity of fluazinam with His9‐MBP‐TEV‐ATPh‐D42A, D42H, F43A, and F43S was reduced to varying degrees. Mutations at residue Asp42 led to a more significant decrease in affinity, with K d values of 639 ± 431 nM for D42A and 1.61 ± 1.36 μM for D42H. CONCLUSION Based on these data, we speculate that fluazinam binds with FfATPh, with Asp42 serving as key binding sites. These findings provide a new molecular perspective on the mode of action of fluazinam and lay the foundation for its precise structural optimization.