Identification and functional characterization of a p-coumaroyl CoA 2′-hydroxylase involved in the biosynthesis of coumarin skeleton from Peucedanum praeruptorum Dunn
- 408 Downloads
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.
- Kai K, Mizutani M, Kawamura N, Yamamoto R, Tamai M, Yamaguchi H, Sakata K, Shimizu B (2008) Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana. Plant J 55:989–999. doi: 10.1111/j.1365-313X.2008.03568.x CrossRefPubMedGoogle Scholar
- Keating GJ, O’Kennedy R (1997) The chemistry and occurrence of coumarins. In: O’Kennedy R, Thornes RD (eds) Coumarins: biology, applications and mode of action. Wiley, Chichester, pp 23–66Google Scholar
- Kim JH, Kim JK, Ahn EK, Ko HJ, Cho YR, Lee CH, Kim YK, Bae GU, Oh JS, Seo DW (2015) Marmesin is a novel angiogenesis inhibitor: regulatory effect and molecular mechanism on endothelial cell fate and angiogenesis. Cancer Lett 369:323–330. doi: 10.1016/j.canlet.2015.09.021 CrossRefPubMedGoogle Scholar
- Liu T, Yao R, Zhao Y, Xu S, Huang C, Luo J, Kong L (2017) Cloning, functional characterization and site-directed mutagenesis of 4-coumarate: coenzyme A ligase (4CL) involved in coumarin biosynthesis in Peucedanum praeruptorum Dunn. Front Plant Sci 8:4. doi: 10.3389/fpls.2017.00004 PubMedPubMedCentralGoogle Scholar
- Matsumoto S, Mizutani M, Sakata K, Shimizu B (2012) Molecular cloning and functional analysis of the ortho-hydroxylases of p-coumaroyl coenzyme A/feruloyl coenzyme A involved in formation of umbelliferone and scopoletin in sweet potato, Ipomoea batatas (L.) Lam. Phytochemistry 74:49–57. doi: 10.1016/j.phytochem.2011.11.009 CrossRefPubMedGoogle Scholar
- Roselli S, Olry A, Vautrin S, Coriton O, Ritchie D, Galati G, Navrot N, Krieger C, Vialart G, Berges H, Bourgaud F, Hehn A (2016) A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip. Plant J 89:1119–1132. doi: 10.1111/tpj.13450 CrossRefGoogle Scholar
- Schlucking K, Edel KH, Koster P, Drerup MM, Eckert C, Steinhorst L, Waadt R, Batistic O, Kudla J (2013) A new β-estradiol-inducible vector set that facilitates easy construction and efficient expression of transgenes reveals CBL3-dependent cytoplasm to tonoplast translocation of CIPK5. Mol Plant 6:1814–1829. doi: 10.1093/mp/sst065 CrossRefPubMedGoogle Scholar
- Schmittgen TD (2006) Quantitative gene expression by real-time PCR: a complete protocol. In: Dorak MT (ed) Real-time PCR. Taylor and Francis Press, New York, pp 127–137Google Scholar
- Shao M (2010) Pharmacopoeia of the People’s Republic of China (Part I). Chemical Industry Press, BeijingGoogle Scholar
- Teutsch HG, Hasenfratz MP, Lesot A, Stoltz C, Garnier JM, Jeltsch JM, Durst F, Werck-Reichhart D (1993) Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway. Proc Natl Acad Sci USA 90:4102–4106CrossRefPubMedPubMedCentralGoogle Scholar
- Vialart G, Hehn A, Olry A, Ito K, Krieger C, Larbat R, Paris C, Shimizu B, Sugimoto Y, Mizutani M, Bourgaud F (2012) A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2′-hydroxylase activity (C2′H): a missing step in the synthesis of umbelliferone in plants. Plant J 70:460–470. doi: 10.1111/j.1365-313X.2011.04879.x CrossRefPubMedGoogle Scholar
- Witaicenis A, Seito LN, da Silveira Chagas A, de Almeida LD Jr, Luchini AC, Rodrigues-Orsi P, Cestari SH, Di Stasi LC (2014) Antioxidant and intestinal anti-inflammatory effects of plant-derived coumarin derivatives. Phytomedicine 21:240–246. doi: 10.1016/j.phymed.2013.09.001 CrossRefPubMedGoogle Scholar
- Zhao Y, Liu T, Luo J, Zhang Q, Xu S, Han C, Xu J, Chen M, Chen Y, Kong L (2015) Integration of a decrescent transcriptome and metabolomics dataset of Peucedanum praeruptorum to investigate the CYP450 and MDR genes involved in coumarins biosynthesis and transport. Front Plant Sci 6:996. doi: 10.3389/fpls.2015.00996 PubMedPubMedCentralGoogle Scholar
- Zhao Y, Luo J, Xu S, Wang W, Liu T, Han C, Chen Y, Kong L (2016a) Selection of reference genes for gene expression normalization in Peucedanum praeruptorum Dunn under abiotic stresses, hormone treatments and different tissues. PLoS ONE 11:e0152356. doi: 10.1371/journal.pone.0152356 CrossRefPubMedPubMedCentralGoogle Scholar
- Zobel AM (1997) Coumarins in fruits and vegetables. In: Tomas-Barberan FAA, Robins RJ (eds) Phytochemistry of fruits and vegetables. Clarendon Press, Oxford, pp 173–203Google Scholar