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.

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

“…Construction of GFP fusion with the N terminal region of Pp4CLs and the transient expression in Arabidopsis protoplasts were conducted according to previously described protocol (Zhao et al, 2016). For amplification of Pp4CLs (Pp4CL1 and Pp4CL10), primers were designed and listed in Supplementary Table S1.…”

“…To unveil the routes for biosynthesis of the coumarins, we previously examined the transcriptome of P. praeruptorum and used transcriptomics in combination with metabolomics to discover candidate genes that participated in the biosynthesis and transport of coumarins (Zhao et al, 2015). Subsequently, we biochemically characterized the BMT responsible for the formation of coumarins in P. praeruptorum (Zhao et al, 2016). Here, we describe the cloning of three novel full-length cDNAs encoding 4CL and enzymatic characterization of Pp4CL1 involved in the biosynthesis of coumarins in P. praeruptorum using Escherichia coli recombinant proteins.…”

Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants

“…Other P450 genes involved in subsequent modifications of the FC cores include CYP71AZ4 , which encodes a psoralen 8‐hydroxylase (Krieger et al ., ), and CYP71AZ1/6 , which encodes a xanthotoxin 5‐hydroxylase (Krieger et al ., ). Bergaptol 5‐ O ‐methyltransferases have also been reported to belong to the SABATH superfamily (Hehmann et al ., ; Ishikawa et al ., ; Zhao et al ., ) (Fig. ).…”

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

“…O -methylation is one of the most important reactions in coumarin biosynthesis; it creates a series of structurally modified compounds (e.g., xanthotoxin, bergapten, scopoletin, and isoscopoletin, Figure S1) [9,10,11]. However, little is known about the methylation mechanism of coumarin derivatives [9,12,13].…”

“…O -methylation is one of the most important reactions in coumarin biosynthesis; it creates a series of structurally modified compounds (e.g., xanthotoxin, bergapten, scopoletin, and isoscopoletin, Figure S1) [9,10,11]. However, little is known about the methylation mechanism of coumarin derivatives [9,12,13]. Moreover, despite the first successful cloning of coumarin-specific bergaptol O -methylation (BMT) in 2004, there is currently no information regarding how esculetin and other hydroxylated coumarins are O -methoxylated (Figure S1) [11,14].…”