Redesigning a One‐Phase Enzymatic Assay to Streamline High‐Throughput Screening of Phytoene Desaturase Inhibitors

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Phytoene desaturase (PDS) is an important target for the development of bleaching herbicides. The lack of the simple and efficient method to directly measure PDS enzyme activity has been the major hurdle to in vitro screening of potent PDS inhibitors. Here, we propose to substitute the traditionally used two‐phase reaction including water and organic solvents with the one‐phase enzymatic reaction as a simple and efficient method to determine PDS enzyme activity. Through combining the fast substrate extraction and high‐performance liquid chromatography, the K m and V max of the Syn PDS enzyme from Synechococcus elongatus was calculated as 24.6 µM and 0.06 µM/min, respectively. Furthermore, the inhibitory activity of seven commercial PDS herbicides was also determined, which was consistent with the molecular docking results. This one‐phase‐based in vitro PDS enzyme activity assay method effectively improved the substrate‐enzyme interaction and reduce the adverse effect of organic solvent on enzyme activity. It will provide a fast and direct evaluation method for the high‐throughput screen of potent PDS inhibitors rather than using the protein‐binding assay.