An in vitro screening assay to discover novel inhibitors of 4-coumarate:CoA ligase |
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| Authors: | Yun Min-Soo Chen Weijun Deng Fan Kiyokawa Takahiro Mametsuka Koki Yogo Yasuhiro |
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| Institution: | Upland Weed Laboratory, Department of Field Environment, National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki, Japan. msyun@nias.affrc.go.jp |
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| Abstract: | 4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) exists only in plants and plays an important role in the phenylpropanoid pathway. Identification of inhibitors targeting 4CL provides a novel approach for developing effective plant growth inhibitors (PGIs). The full-length gene of tobacco 4CL (Nt4CL1) was cloned and expressed in Escherichia coli Cast & Chalm. The recombinant 4CL protein was extracted and purified by several purification steps including gel-filtration and anion-exchange chromatography. 4CL activity assay was miniaturized and optimized using a 96-well microplate and a reader. Among 28 existing herbicides, propanil and swep strongly inhibited in vitro 4CL enzyme activity, and they were selected for further studies. The process of this assay can be developed into a high-throughput screening system of PGI targeting 4CL in the phenylpropanoid pathway. |
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| Keywords: | 4‐coumarate:CoA ligase high‐throughput screening plant growth inhibitors propanil swep |
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