Dataset Information

Cloning, Functional Characterization, and Catalytic Mechanism of a Bergaptol O-Methyltransferase from Peucedanum praeruptorum Dunn.

ABSTRACT: Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin, and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum transcriptome database using the nucleotide sequence of Ammi majus bergaptol O-methyltransferase (AmBMT, GenBank accession No: AY443006) as a template. As a result, a 1335 bp full-length of cDNA sequence which contains an open reading frame of 1080 bp encoding a BMT polypeptide of 359 amino acids was obtained. The recombinant protein was functionally expressed in Escherichia coli and displayed an observed activity to bergaptol. In vitro experiments show that the protein has narrow substrate specificity for bergaptol. Expression profile indicated that the cloned gene had a higher expression level in roots and can be induced by methyl jasmonate (MeJA). Subcellular localization analysis showed that the BMT protein was located in cytoplasm in planta. Homology modeling and docking based site-directed mutagenesis have been employed to investigate the amino acid residues in BMT required for substrate binding and catalysis. Conservative amino acid substitutions at residue H264 affected BMT catalysis, whereas substitutions at residues F171, M175, D226, and L312 affected substrate binding. The systemic study summarized here will enlarge our knowledge on OMTs and provide useful information in investigating the coumarins biosynthesis mechanism in P. praeruptorum.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC4879325 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cloning, Functional Characterization, and Catalytic Mechanism of a Bergaptol O-Methyltransferase from Peucedanum praeruptorum Dunn.

Zhao Yucheng Y Wang Nana N Zeng Zhixiong Z Xu Sheng S Huang Chuanlong C Wang Wei W Liu Tingting T Luo Jun J Kong Lingyi L

Frontiers in plant science 20160525

Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin, and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum ...[more]

PMID: 27252733

Similar Datasets

Project description:Peucedanum praeruptorum Dunn is a commonly used traditional Chinese medicine that is abundant in furano- and dihydropyrano coumarins. When P. praeruptorum reaches the bolting stage, the roots gradually lignified, and the content of coumarins declines rapidly. Non-bolting has always been a decisive factor for harvesting the P. praeruptorum materials. To evaluate the amount of coumarin components in unbolted and bolted P. praeruptorum, the variations of praeruptorin A, praeruptorin B, praeruptorin E, peucedanocoumarin I, and peucedanocoumarin II were determined. Additionally, 336,505 transcripts were obtained from the comparative transcriptome data. Among them, a total of 1,573 differentially expressed genes were screened out. To identify the critical genes involved in coumarin biosynthesis, comparative transcriptomics coupled with co-expression associated analysis was conducted. Finally, coumarin biosynthesis-related eighteen candidate genes were selected for the validation of qPCR. Additionally, a phylogenetic tree and the expression profile of ATP-binding cassette (ABC) transporters were constructed. To clarify the main genes in the regulation of coumarin biosynthesis, the interaction network of the co-expression genes from thirteen modules was constructed. Current results exhibited the significant increment of praeruptorin A, praeruptorin B and praeruptorin E in the bolted P. praeruptorum. Although, peucedanocoumarin I and peucedanocoumarin II were slightly increased. Besides the content of coumarins, the essential genes involved in the coumarin biosynthesis also exhibited an overall downward trend after bolting. Three peroxidases (PRXs) involved in the production of lignin monomers had been demonstrated to be downregulated. PAL, C4H, HCT, COMT, CCoAOMT, and some ABC transporters were dramatically downregulated at the bolting stage. These results indicated that the downregulation of coumarin biosynthetic genes in the bolted P. praeruptorum ultimately reduced the formation of coumarins. However, the mechanism through which bolting indirectly affects the formation of coumarin still needs extra functional verification.

Project description:BackgroundPeucedanum praeruptorum Dunn, a traditional Chinese herbal medicine, contains coumarin and volatile oil components that have clinical application value. However, early bolting often occurs in the medicinal materials of Apiaceae plants. The rhizomes of the medicinal parts are gradually lignified after bolting, resulting in a sharp decrease in the content of coumarins. At present, the link between coumarin biosynthesis and early bolting in P. praeruptorum has not been elucidated.ResultsCombining the genome sequencing and the previous transcriptome sequencing results, we reanalyzed the differential transcripts of P. praeruptorum before and after bolting. A total of 62,088 new transcripts were identified, of which 31,500 were unknown transcripts. Functional classification and annotation showed that many genes were involved in the regulation of transcription, defense response, and carbohydrate metabolic processes. The main domains are the pentatricopeptide repeat, protein kinase, RNA recognition motif, leucine-rich repeat, and ankyrin repeat domains, indicating their pivotal roles in protein modification and signal transduction. Gene structure analysis showed that skipped exon (SE) was the most dominant alternative splicing, followed by the alternative 3' splice site (A3SS) and the alternative 5' splice site (A5SS). Functional enrichment of differentially expressed genes showed that these differentially expressed genes mainly include transmembrane transporters, channel proteins, DNA-binding proteins, polysaccharide-binding proteins, etc. In addition, genes involved in peroxisome, hexose phosphate pathway, phosphatidylinositol signaling system, and inositol phosphate metabolism pathway were greatly enriched. A protein-protein interaction network analysis discoverd 1,457 pairs of proteins that interact with each other. The expression levels of six UbiA genes, three UGT genes, and four OMT genes were higher during the bolting stage. This observation suggests their potential involvement in the catalytic processes of prenylation, glycosylation, and methylation of coumarins, respectively. A total of 100 peroxidase (PRX) genes were identified being involved in lignin polymerization, but only nine PRX genes were highly expressed at the bolting stage. It is worth noting that 73 autophagy-related genes (ATGs) were first identified from the KEGG pathway-enriched genes. Some ATGs, such as BHQH00009837, BHQH00013830, and novel8944, had higher expression levels after bolting.ConclusionsComparative transcriptome analysis and large-scale genome screening provide guidance and new opinions for the identification of bolting-related genes in P. praeruptorum.

Dataset Information

Cloning, Functional Characterization, and Catalytic Mechanism of a Bergaptol O-Methyltransferase from Peucedanum praeruptorum Dunn.

Publications

Cloning, Functional Characterization, and Catalytic Mechanism of a Bergaptol O-Methyltransferase from Peucedanum praeruptorum Dunn.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets