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.

Keywords: O-methyltransferase (OMT); Peucedanum praeruptorum; biosynthesis mechanisms; coumarins; docking.

FIGURE 1

Schematic outline of OMTs involved coumarins biosynthesis. O-MT, O-methyl-transferase; XMT, Xanthotoxiol O-methyltransferase; BMT, Bergaptol O-methyltransferase. The oval in blue is the reaction investigated in this study.

FIGURE 2

Sequence alignment of PpBMT with other plant OMTs. Boxed amino acids (regions I–V) represent conserved regions. Residues involved in SAM binding, substrate binding, catalytic center are indicated by boxes, lines and pentagram, respectively. Accession numbers: AmBMT (AY443006), HvOMT (CAA54616), MsChOMT (AAB48059), MsCOMT (AAB46623).

FIGURE 3

Phylogenetic relationships between PpBMT and other OMTs. Neighbor-joining phylogram for amino acid sequences of PpBMT and other OMTs were drawn by ClustalW, showing the result of 1000 bootstrap tests using MEGA5 software. Accession numbers: AmBMT (Ammi majus AY443006), GlBMT (Glehnia littoralis AB363638), AdBMT (Angelica dahurica AEO21927.1), MsChOMT (M. sativa AAB48059), MsCOMT (M. sativa AAB46623), HvOMT (Hordeum vulgare CAA54616), AtOMT (Arabidopsis NP_195242.1), CaOMT (Capsicum annuum Q9FQY8.2), NtOMT (Nicotiana tabacum CAA52461.1), CaFOMT (Chrysosplenium americanum Q42653.1), CbIEMT (Clarkia breweri O04385.2), EgCOMT (Eucalyptus gunnii P46484.1), CjOMT (Coptis japonica Q39522.1), PsOMT (Pisum sativum O24305.1), PdCOMT (Prunus dulcis Q43609.1), PpOMT (Pyrus pyrifolia BAA86059.1), MsIOMT (M. sativa O22309.1), ObCOMT (Ocimum basilicum Q9XGW0.1), PrOMT (Pinus radiata AAD24001.1), PaOMT (Prunus armeniaca AAB71213.1), TaOMT (Triticum aestivum AAD10485.1), ZvCOMT (Zinnia violacea Q43239.1).

FIGURE 4

Enzymatic characteristics of PpBMT expressed in E. coli. (A) E. coli without transform. (B) E. coli containing empty vector pET28a. (C) E. coli containing recombinant plasmid pET28a-BMT. (D) MS fragmentation of substrate. (E) MS fragmentation of products. Ultraviolet absorption spectra and the structure of substrate and product are also shown beside the corresponding peak.

FIGURE 5

Substrate specificity of recombinant PpBMT. The activity of the enzyme toward bergaptol is set as 100% and the structure of candidate substrates are drawn in the picture. N.D. represents no detectable.

FIGURE 6

Expression profiles of PpBMT. (A) Tissue-specific expression. (B) Gene expression after MeJA treatment. R, roots; S, stems; L, leaves. 0, 1, 3, 6, 9, 12, and 24 h represent the time interval after MeJA treatment. The expression level of leaves in (A) and 0 h in (B) were set as reference in each group. Each bar represents the mean value results from the mean of triplicate experiments ±SD.

FIGURE 7

Confocal fluorescence microscopy images of PpBMT-GFP fusion protein in transgenic Arabidopsis protoplasts. Control, Arabidopsis protoplasts without be transformed any vectors. 35S::GFP, Arabidopsis protoplasts contains empty vector. 35S::PpBMT-GFP, Arabidopsis protoplasts contains constructed pCAMBIA-1302-BMT plasmid. The photographs were taken in the red channel (Left, chlorophyll), in the blue channel (Middle, GFP) and in their combination (Right, merge). Auto-fluorescence of chlorophylls in chloroplast was used as a control for plastid targeting. The images were obtained at 16 h of transformation. Scale bars show 10 μm.

FIGURE 8

Homology modeling and docking of PpBMT with SAM and bergaptol. (A) Three-dimensional model of PpBMT-SAM. (B) Three-dimensional model of PpBMT-bergaptol. (C) Two-dimensional model of PpBMT-SAM. (D) Two-dimensional model of PpBMT-bergaptol. (E) Site-directed mutagenesis of PpBMT according to the docking results. Protein was depicted in line and the substrate was shown in spheres. The activity of PpBMT was set as reference. Each bar represents the mean value results from the mean of triplicate experiments ±SD.