Identification and functional characterization of a p-coumaroyl CoA 2′-hydroxylase involved in the biosynthesis of coumarin skeleton from Peucedanum praeruptorum Dunn
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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.
Keywordsp-Coumaroyl CoA 2′-hydroxylase Peucedanum praeruptorum Coumarin biosynthesis Functional characterization Site-directed mutagenesis
This research was supported in part by the Natural Science Fund in Jiangsu Province (BK20170736), China Postdoctoral Science Foundation (1600020005), the National Natural Science Foundation of China (81430092), the Program for New Century Excellent Talents in University (NCET-2013-1035), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), and the Ph.D. Programs Foundation of Ministry of Education of China (20120096130002). We also thank the Cellular and Molecular Biology Center of China Pharmaceutical University for assistance with confocal microscopy work and we are grateful to Xiao-Nan Ma for her technical help.
RY, YZ, JL and LK conceived and designed the work; RY, TL, YZ, SX and ZS performed the experiments; RY and CH interpreted and analyzed the data; RY wrote the paper; RY, YZ, TL, JL and LK revised the paper critically. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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