Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.
Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.
复制标题
白花前胡香豆素合成相关酶4-香豆酸：辅酶A连接酶（4CL）的克隆、功能分析及定点突变
DOI：
10.3389/fpls.2017.00004
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发表时间：
2017
影响因子：
5.6
通讯作者：
Kong L
中科院分区：
文献类型：
--
作者：
Liu T;Yao R;Zhao Y;Xu S;Huang C;Luo J;Kong L
香豆素是常见中药材前胡中的主要生物活性成分。然而，目前尚未鉴定出参与 P. praeruptorum 香豆素核心结构生物合成的基因。 4-香豆酸：CoA 连接酶 (4CL) 催化羟基肉桂酸 CoA 酯的形成，并在从一般苯丙素代谢到香豆素主要分支途径的分叉点发挥重要作用。在此，从 P. praeruptorum 中分离出三个新的推定 4CL 基因（Pp4CL1、Pp4CL7 和 Pp4CL10）。重组蛋白的生化表征表明，Pp4CL1 利用对香豆酸和阿魏酸作为 P. praeruptorum 中香豆素生物合成的两种主要底物。此外，Pp4CL1 还表现出对咖啡酸、肉桂酸、异阿魏酸和邻香豆酸的活性，代表真正的 4CL。 Pp4CL7和Pp4CL10对羟基肉桂酸化合物没有催化活性。但它们与真正的 4CL 具有密切的系统发育关系，并被定义为 4CL 样基因。在所有假定的4CL中，Pp4CL1是根中表达量最高的基因，与幼苗相比，其在成熟根中的表达水平显着上调。亚细胞定位研究表明 Pp4CL1 和 Pp4CL10 蛋白定位于细胞质中。此外，Pp4CL1 的定点诱变证明 Tyr-239、Ala-243、Met-306、Ala-309、Gly-334、Lys-441、Gln-446 和 Lys-526 氨基酸对于底物结合或催化活性至关重要。 Pp4CL1 的表征和定点诱变研究为阐明 P. praeruptorum 中香豆素的生物合成机制奠定了坚实的基础，并为理解这一重要蛋白质家族的结构-功能关系提供了进一步的见解。
Coumarins are the main bioactive compounds in Peucedanum praeruptorum Dunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin in P. praeruptorum have 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 from P. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilized p-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis in P. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, and o-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 in P. praeruptorum and provides further insights in understanding the structure–function relationships of this important family of proteins.