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An in vitro screening assay to discover novel inhibitors of 4‐coumarate:CoA ligase
Author(s) -
Yun MinSoo,
Chen Weijun,
Deng Fan,
Kiyokawa Takahiro,
Mametsuka Koki,
Yogo Yasuhiro
Publication year - 2006
Publication title -
pest management science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.296
H-Index - 125
eISSN - 1526-4998
pISSN - 1526-498X
DOI - 10.1002/ps.1277
Subject(s) - dna ligase , biochemistry , enzyme , high throughput screening , phenylpropanoid , biology , escherichia coli , in vitro , recombinant dna , biosynthesis , gene
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. Copyright © 2006 Society of Chemical Industry