Structurally diverse herbicides inhibit β-keto-acyl-CoA synthases from monocotyledonous and dicotyledonous plant species.

Very-long-chain fatty acids (VLCFA) serve as precursors for various lipids that play crucial physiological and structural roles in plants. The β-keto-acyl-CoA synthase (KCS) catalyzes the initial step of VLCFA elongation and determines the chain-length substrate specificity of the fatty acid elongase complex. Following an extensive phylogenetic analysis, 28 KCS genes from various plant species, including 17 functionally uncharacterized genes, were expressed in Saccharomyces cerevisiae, and their VLCFAs profiles were analyzed using gas chromatography-mass spectrometry. Yeast expressing active KCS were subsequently exposed in vivo to various Group 15 herbicides to investigate their mode of action and selectivity. These experiments revealed differential sensitivities of KCS enzymes to benfuresate, cafenstrole, flufenacet, pyroxasulfone, and metazachlor, while endogenous VLCFA synthesis in yeast, which relies on Elop-type elongases, remained unaffected. Furthermore, to address a significant gap in understanding the inhibitory potency of Group 15 herbicides, we developed an in vitro enzymatic assay based on the quantification of the 3-oxo-products generated during the KCS-catalyzed reaction using high-performance liquid chromatography-mass spectrometry coupled with UV detection. This novel biochemical approach enabled the evaluation of inhibitor potency as well as the kinetic characterization of both established and newly identified KCS enzyme inhibitors.