An in vitro screening assay to discover novel inhibitors of 4-coumarate:CoA ligase.
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.
Yun
Min-Soo
MS
Upland Weed Laboratory, Department of Field Environment, National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki, Japan. msyun@nias.affrc.go.jp
Chen
Weijun
W
Deng
Fan
F
Kiyokawa
Takahiro
T
Mametsuka
Koki
K
Yogo
Yasuhiro
Y
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
D006540:Herbicides; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase