Basing target enzyme study the enantioselective bioactivity action mechanism of flusulfinam, a novel HPPD inhibitor herbicide

Flusulfinam is a novel chiral amide herbicide widely used for controlling annual weeds in rice paddies. However, the mechanism underlying their enantioselective herbicidal activity remain unclear. Herein, it was found that flusulfinam enantiomers, similar to typical HPPD inhibitor mesotrione, reduced chlorophyll and carotenoid levels, decreased HPPD enzyme activity, and upregulated gene expression. Additionally, homogentisate supplementation alleviated the bleaching symptoms caused by flusulfinam and all these results validate that flusulfinam is indeed an HPPD inhibitor. To further investigate the mechanism of enantioselectivity, molecular docking was used and showed that R-flusulfinam (-6.55 kcal/mol) had higher binding energy than S-flusulfinam (-5.60 kcal/mol), due to more stable hydrogen bonds with Gln293. After mutating Gln293 to His, the IC₅₀ values for R-flusulfinam and S-flusulfinam on MutQ293H were 0.73 mg/L and 0.11 mg/L, respectively, indicating swapped enantioselective inhibition compared to AtHPPD, with IC₅₀ values of 0.52 mg/L and 1.93 mg/L, respectively. The Microscale Thermophoresis assay further revealed that the dissociation constant (Kd) for MutQ293H with R-flusulfinam was 20.40 ± 4.19 μM, similar to the Kd value of 15.63 ± 4.51 μM for S-flusulfinam. The findings reveal that mutation of the Gln293 residue in the AtHPPD enzyme significantly altered its enantioselective inhibition by flusulfinam. This study is the first to verify the mode of action of flusulfinam and identifies that Gln293 may play a key role in flusulfinam enantioselectivity in the AtHPPD, laying the foundation for future HPPD inhibitor development based on flusulfinam.