Catalytic Promiscuity Underpins Metabolic Resistance to Auxinic Herbicides in <i>Echinochloa phyllopogon</i>

High Resolution Image Download MS PowerPoint Slide Auxinic herbicides remain essential for weed control, yet metabolic resistance to these herbicides is poorly understood. Here we show that catalytically promiscuous CYP81A P450s from Echinochloa phyllopogon detoxify auxinic herbicides. Arabidopsis thaliana expressing CYP81A12 or CYP81A21 showed decreased sensitivity to quinclorac. In an Escherichia coli whole-cell assay, these enzymes produced a putative hydroxylated quinclorac metabolite. Docking and 300 ns molecular dynamics simulations indicated that active CYP81As maintain quinclorac in a productive pose close to the heme, with pocket geometry and water exclusion enabling precise substrate–heme alignment, whereas the inactive CYP81A18 retains quinclorac farther from the catalytic center. Consistent with resistant E. phyllopogon, transgenic Arabidopsis expressing CYP81A12 or CYP81A21 produced less ethylene after quinclorac treatment, linking detoxification to suppressed auxinic phytotoxic responses. CYP81As also conferred resistance to florpyrauxifen-benzyl and 2,4-D, highlighting cross-resistance across auxinic chemistries. These findings reveal a structural basis for CYP81A-mediated catalytic promiscuity underlying metabolic resistance to auxinic herbicides.