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

Abstract

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.

Tissue-Specific Transcriptome Analysis Reveals Candidate Genes for Terpenoid and Phenylpropanoid Metabolism in the Medicinal PlantFerula assafoetida

“…The coumarin umbelliferone and the flavone luteolin are commonly occurring phenylpropanoid metabolites in members of the Apiaceae family and serve as a precursor for a range of specialized metabolites, including pyrano-coumarins and furano-coumarins (Luo et al 2017; Yao et al 2017). Therefore, we quantified umbelliferone and luteolin across the select plant organs (Supplementary Figure S7A and S7B) and isolated oleo-gum-resin fractions (Supplementary Figure S7C).…”

“…Hence, a more expansive evolutionary divergence of this pathway component may have occurred in F. assafoetida . In contrast, only a single transcript was identified that showed significant similarity to p-coumaroyl-CoA 2′-hydroxylase (C2’H) enzymes that catalyze the hydroxylation of the 4CL product p-coumaroyl-CoA as a key reaction in the formation of coumarins such as umbelliferone (Yao et al 2017) (Figure 3). Unlike coumarins, biosynthesis of flavone metabolites, including luteolin abundant in F. assafoetida oleo-gum-resin and other organs (Figure S7), proceeds through the activity of a chalcone synthase (CHS), followed by further modifications by chalcone isomerases (CHI), flavone synthases (FNS), and flavanone 3-hydroxylases (F3′H) (Bourgaud et al 2006; Naoumkina et al 2010; Vogt 2010).…”

The Molecular and Structural Basis of O-methylation Reaction in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn

“…Then, the fragment was ligated into the expression vector pET28a for protein expression and purification. For COMT-S purification, 6× His purification label according to our previously report was used [26]. The protein was further purified via gel-filtration chromatography on a Superdex 200 column (GE Healthcare, Pittsburgh, PA, USA) equilibrated with running buffer (RB) (25 mM Tris-HCl pH 8.0, 150 mM NaCl).…”

Engineering Plant Secondary Metabolism in Microbial Systems

“…flavonoids and stilbenoids, coumarins are not polyketides, as their lactone ring structure is derived from hydroxylation, isomerization, and lactonization of phenolic acids (cinnamic, p-coumaric, caffeic, or ferulic acid) or the corresponding phenolic acyl-CoA esters (Kai et al, 2008;Yao et al, 2017).…”