Identification and functional characterization of a p-coumaroyl CoA 2'-hydroxylase involved in the biosynthesis of coumarin skeleton from Peucedanum praeruptorum Dunn.

KEY MESSAGE: A p-coumaroyl CoA 2'-hydroxylase responsible for the formation of coumarin lactone ring was identified from Peucedanum praeruptorum Dunn and functionally characterized in vitro. Coumarins are important plant secondary metabolites with a variety of biological activities. Ortho-hydroxylation of cinnamates leads to the formation of coumarin lactone ring and is generally thought to be a key step in coumarin biosynthesis. However, ortho-hydroxylases, especially p-coumaroyl CoA 2'-hydroxylase (C2'H) responsible for the biosynthesis of the most common coumarin skeleton, have received insufficient attention. Here, a putative ortho-hydroxylase PpC2'H was isolated from P. praeruptorum Dunn, a traditional Chinese medicinal herb rich in coumarins. Expression profile indicated that PpC2'H exhibited the highest transcript level in roots and could be up-regulated by MeJA elicitation. Subcellular localization of PpC2'H was demonstrated to be cytosol in planta. In order to functionally characterize PpC2'H, the purified recombinant protein was incubated with various potential substrates. HPLC-ESI-MS analysis indicated that PpC2'H catalyzed the conversion of p-coumaroyl CoA into hydroxylated intermediate, which then underwent spontaneous lactonization to generate umbelliferone. Our data also showed that light would promote the spontaneous process. In addition, based on homology modeling and site-directed mutagenesis, amino acid residues Phe-130, Lys-141, Asn-207, His-224, Asp-226, His-282 and Phe-298 were verified essential for enzymatic activity. These findings provide insight into structure-function relationship of this pivotal ortho-hydroxylase and also contribute to elucidating the biosynthetic mechanism of coumarin skeleton.

Functional characterization and correlation analysis of phenylalanine ammonia-lyase (PAL) in coumarin biosynthesis from Peucedanum praeruptorum Dunn.

Coumarins exhibit many biological activities and are the main specialised metabolites of Peucedanum praeruptorum Dunn, an important plant used in traditional Chinese medicine. In preliminary studies, we cloned several genes involved in coumarin biosynthesis in P. praeruptorum, such as 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hydroxylase (C2'H), feruloyl CoA 6'-hydroxylase (F6'H) and bergaptol O-methyltransferase (BMT). However, phenylalanine ammonia-lyase (PAL) in P. praeruptorum (PpPAL) has not yet been studied. In the present study, we cloned one novel PpPAL gene. Subsequently, the relationship between gene and compounds was studied using quantitative real-time PCR (qRT-PCR) and High Performance Liquid Chromatography (HPLC) analysis. Then, enzyme function was analyzed with L-phenylalanine (L-Phe) as substrate. These experiments showed that the coumarin content could be upregulated by methyl jasmonate (MeJA), UV irradiation and cold, which was consistent with increased expression levels of PpPAL. In addition, correlation analysis indicated that coumarins were partially related to PpPAL. And the recombinant protein could catalyze the conversion of L-Phe to trans-cinnamic acid (t-CA) with a Km of 120 ±â€¯33 µM and a Kcat of 117 ±â€¯32 min-1. Besides, Tyr110, Phe116, Gly117, Ser206, Leu209, Leu259, Tyr354, Arg357, Asn387 and Phe403 were essential for enzymatic activity based on three-dimensional modeling and site-directed mutagenesis experiments. Altogether these results highlight the importance of PpPAL in abiotically induced coumarin biosynthesis and provide further insights regarding the structure-function relationships of this protein.

Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.

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.