title: An in vitro screening assay to discover novel inhibitors of 4-coumarate:CoA ligase.
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.
authors.affiliation: Upland Weed Laboratory, Department of Field Environment, National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki, Japan. msyun@nias.affrc.go.jp
authors.forename: Min-Soo
authors.initials: MS
authors.lastname: Yun
authors.forename: Weijun
authors.initials: W
authors.lastname: Chen
authors.forename: Fan
authors.initials: F
authors.lastname: Deng
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kiyokawa
authors.forename: Koki
authors.initials: K
authors.lastname: Mametsuka
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yogo
mesh_terms: D000595:Amino Acid Sequence; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006540:Herbicides / Q000737:chemistry*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics
chemical_list: D006540:Herbicides; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase