Uncovering the Differed Susceptibility of Fusarium oxysporum (Fo32931 and FocII5) to Fungicide Phenamacril: From Computational and Experimental Perspectives

Fo32931 and FoCII5 are two subtypes of Fusarium oxysporum (Fo), a pathogenic filamentous fungus. Phenamacril (PHA), a Fusarium-specific fungicide that targets myosin I, exhibits significant hyphal growth inhibition in Fo32931 but shows weak resistance in FocII5, despite only two amino acid differences in the PHA-binding pocket of myosin I. In this study, we aim to elucidate the molecular basis for the differential sensitivity ofF. oxysporum myosin I variants (FoMyoI³²⁹³¹ and FoMyoI^cII5) to phenamacril through computational methods and biochemical validation. The results suggest that phenamacril functions as an allosteric inhibitor for FoMyoI³²⁹³¹, inhibiting the large oscillation of the converter lever domain (CLD) upon ATP binding and promoting the opening of the outer cleft, further impairing protein function. PHA significantly reduced the coupling between the CLD, especially the converter, and the catalytic center, diminishing the response of the CLD to the motor domain in FoMyoI³²⁹³¹. From the residue mutation experiment, we found that the S418T substitution in FoMyoI^cII5 is the key to the reduced phenamacril sensitivity of FocII5. According to the microscale thermophoresis (MST) assay and pocket conformation analysis, the S418T mutation disturbs the orientation of pocket residues, especially Lys537, leading to a looser pocket and reduced interaction between Lys537 and phenamacril, which lowers the binding affinity of FoMyoI^cII5 for phenamacril. These findings provide deeper insights into the reasons for the lower sensitivity of FoCII5 to phenamacril from both molecular and structural perspectives and will also guide the design of novel inhibitors against resistant Fusarium spp., like FoCII5.