Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC values: 6.47 μM for SmM, 3.58 μM for NM2a, 2.30 μM for NM2b, and 1.57 μM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.

Phenamacril is a Fusarium -specific fungicide used for Fusarium head blight management. The target of phenamacril is FgMyo1, the sole class I myosin in Fusarium graminearum . The point mutation S217L in FgMyo1 is responsible for the high resistance of F. graminearum to phenamacril. Recent structural studies have shown that phenamacril binds to the 50 kDa cleft of the FgMyo1 motor domain, forming extensive interactions, including a hydrogen bond between the cyano group of phenamacril and the hydroxyl group of S217. Here, we produced FgMyo1 IQ2 , a truncated FgMyo1 composed of the motor domain and two IQ motifs complexed with the F. graminearum calmodulin in insect Sf9 cells. Phenamacril potently inhibited both the basal and the actin-activated ATPase activities of FgMyo1 IQ2 , with an IC 50 in a micromolar range. S217 mutations of FgMyo1 IQ2 substantially increased the IC 50 of phenamacril. S217T or S217L each increased the IC 50 of phenamacril for ∼60-fold, while S217A only increased the IC 50 for ∼4-fold. These results indicate that the hydroxyl group of S217 plays an important, but nonessential role in phenamacril binding and that the bulky side chain at the position 217 sterically hinders phenamacril binding. On the other hand, S217P, which might alter the local conformation of the phenamacril-binding site, completely abolished the phenamacril inhibition. Because the cyano group of phenamacril does not form discernible interactions with FgMyo1 other than the nonessential hydrogen bond with the S217 hydroxyl group, we propose the cyano group of phenamacril as a key modification site for the development of novel fungicides.