title: Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis inPeucedanum praeruptorumDunn.
abstract: Coumarins are the main bioactive compounds inPeucedanum praeruptorumDunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin inP. praeruptorumhave not been identified yet. 4-Coumarate: CoA ligase (4CL) catalyzes the formation of hydroxycinnamates CoA esters, and plays an essential role at the divergence point from general phenylpropanoid metabolism to major branch pathway of coumarin. Here, three novel putative 4CL genes (Pp4CL1, Pp4CL7, and Pp4CL10) were isolated fromP. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilizedp-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis inP. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, ando-coumaric acids and represented a bona fide 4CL. Pp4CL7 and Pp4CL10 had no catalytic activity toward hydroxycinnamic acid compounds. But they had close phylogenetic relationship to true 4CLs and were defined as 4CL-like genes. Among all putative 4CLs, Pp4CL1 was the most highly expressed gene in roots, and its expression level was significantly up-regulated in mature roots compared with seedlings. Subcellular localization studies showed that Pp4CL1 and Pp4CL10 proteins were localized in the cytosol. In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins inP. praeruptorumand provides further insights in understanding the structure-function relationships of this important family of proteins.
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University Nanjing, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University Nanjing, China.
authors.forename: Ruolan
authors.initials: R
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University Nanjing, China.
authors.forename: Yucheng
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Institute of Botany, Jiangsu Province and Chinese Academy of Sciences Nanjing, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University Nanjing, China.
authors.forename: Chuanlong
authors.initials: C
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University Nanjing, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University Nanjing, China.
authors.forename: Lingyi
authors.initials: L
authors.lastname: Kong
keywords: 4-coumarate: CoA ligase; Peucedanum praeruptorum; biochemical characterization; biosynthesis mechanism; site-directed mutagenesis