Bergaptol O-methyltransferase Research Articles

Characterization of Abscisic Acid and Ethylene in Regulating the White Blush in Fresh-Cut Carrots.

The surface of fresh-cut carrots is apt to white blush, however the physiological and molecular mechanism for this process is not yet fully understood. In this study, exogenous abscisic acid (ABA) and ethylene separately promoted and inhibited the white-blush formation after three days after treatment, respectively. Metabolome analysis found that white-blush components mainly consist of p-hydroxyphenyl lignin and guaiacyl lignin. Transcriptome analysis found an increase in the whiteness values was consistent with the higher expression of genes encoding O-methyltransferase, trans-anol O-methyltransferase, bergaptol O-methyltransferase, caffeic acid 3-O-methyltransferase, phenylalanine ammonia-lyase, and ferulate-5-hydroxylase, together with the lower expression of genes encoding cinnamic acid 4-hydroxylase caffeoyl-CoA O-methyltransferase and 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase. In conclusion, ABA plays an important role in lignin biosynthesis essential to the formation of white blush in fresh-cut carrots. This is the first report that uncovers the physiological and molecular causes of white blush in fresh-cut carrots, providing a basis for white-blush control in fresh-cut carrots.

Structure-based tailoring of the first coumarins-specific bergaptol O-methyltransferase to synthesize bergapten for depigmentation disorder treatment

Bergapten has long been used in combination with ultraviolet A irradiation to treat depigmentation disorder. However, extremely low bergapten contents in plants and difficulties in synthesizing bergapten have limited its application. Here, we developed an alternative bergapten-production method. We first determined the crystal structures of bergaptol O-methyltransferase from Peucedanum praeruptorum (PpBMT) and the ternary PpBMT–S-adenosyl-L-homocysteine (SAH)–bergaptol complex to identify key residues involved in bergaptol binding. Then, structure-based protein engineering was performed to obtain PpBMT mutants with improved catalytic activity towards bergaptol. Subsequently, a high-activity mutant was used to produce bergapten for pharmacological-activity analysis. Key PpBMT amino acids involved in bergaptol binding and substrate specificity were identified, such as Asp226, Asp246, Ser265, and Val320. Site-directed mutagenesis and biochemical analysis revealed that the V320I mutant efficiently transformed bergaptol to produce bergapten. Pharmacological-activity analysis indicated that bergapten positively affected hair pigmentation in mice and improved pigmentation levels in zebrafish embryos. This report provides the first description of the catalytic mechanism of coumarins-specific O-methyltransferase. The high-activity V320I mutant protein could be used in metabolic engineering to produce bergapten in order to treat depigmentation disorder. This structure–function study provides an alternative synthesis method and important advances for treating depigmentation disorders.

Functional characterization and correlation analysis of phenylalanine ammonia-lyase (PAL) in coumarin biosynthesis from Peucedanum praeruptorum Dunn

Coumarins exhibit many biological activities and are the main specialised metabolites of Peucedanum praeruptorum Dunn, an important plant used in traditional Chinese medicine. In preliminary studies, we cloned several genes involved in coumarin biosynthesis in P. praeruptorum, such as 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2′-hydroxylase (C2′H), feruloyl CoA 6′-hydroxylase (F6′H) and bergaptol O-methyltransferase (BMT). However, phenylalanine ammonia-lyase (PAL) in P. praeruptorum (PpPAL) has not yet been studied. In the present study, we cloned one novel PpPAL gene. Subsequently, the relationship between gene and compounds was studied using quantitative real-time PCR (qRT-PCR) and High Performance Liquid Chromatography (HPLC) analysis. Then, enzyme function was analyzed with L-phenylalanine (L-Phe) as substrate. These experiments showed that the coumarin content could be upregulated by methyl jasmonate (MeJA), UV irradiation and cold, which was consistent with increased expression levels of PpPAL. In addition, correlation analysis indicated that coumarins were partially related to PpPAL. And the recombinant protein could catalyze the conversion of L-Phe to trans-cinnamic acid (t-CA) with a Km of 120 ± 33 μM and a Kcat of 117 ± 32 min−1. Besides, Tyr110, Phe116, Gly117, Ser206, Leu209, Leu259, Tyr354, Arg357, Asn387 and Phe403 were essential for enzymatic activity based on three-dimensional modeling and site-directed mutagenesis experiments. Altogether these results highlight the importance of PpPAL in abiotically induced coumarin biosynthesis and provide further insights regarding the structure-function relationships of this protein.

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

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.

Constitutive expression of bergaptol O-methyltransferase in Glehnia littoralis cell cultures

We investigated whether exogenously supplied precursors of bergapten, namely umbelliferone, psoralen and bergaptol, could be utilized to produce bergapten without elicitation in Glehnia littoralis cell suspension cultures. The levels of added psoralen and bergaptol in the medium soon decreased, and this was followed by the detection of bergapten in both culture fluid and cells. Umbelliferone was also incorporated but in this case no bergapten was produced; instead, skimmin, umbelliferone monoglucoside, was detected. To determine whether conversion of psoralen to bergapten was due to enzyme induction by precursor feeding, the transcript accumulations and enzyme activities of bergaptol O-methyltransferase (BMT, EC 2.1.1.69), which catalyzes the last step of bergapten synthesis, and of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), which catalyzes the initial step of the phenylpropanoid biosynthetic pathway and is known as a marker enzyme of elicitation, were examined. The results showed that both the expression and the activity of BMT were always detected in all cells, including control cells. Since PAL was slightly induced in the cells supplied with/without precursors, phenylethyl alcohol (PEA, a competitive inhibitor of PAL) was applied to suspension cells prior to the addition of psoralen. PAL activity was effectively inhibited by PEA at 1-5 mM concentrations. Under these conditions, PEA did not affect bergapten production by cell cultures fed with psoralen at all. These results demonstrate that BMT is constitutively expressed in G. littoralis cell cultures.

Computational studies on bergaptol O-methyltransferase from Ammi majus L.: The substrate specificity

In order to understand the mechanisms of substrate specificity and the interaction between bergaptol and bergaptol O-methyltransferase (BMT), a 3D model of BMT is generated based on the crystal structure of caffeic acid 3- O-methyltransferase (COMT EC 2.1.1.68, PDB code 1KYZ) by using the InsightII/Homology module. With the aid of the molecular mechanics and molecular dynamics methods, the final refined model is obtained and its reliability is further assessed by PROCHECK and ProSa2003. With this model, a flexible docking study is performed and the results indicate that BMT has narrow substrate specificity. Although the homology between both proteins is higher than 65% and all amino acids surrounding the binding site, except four residues, are similar in their sequences, the two proteins exhibit different substrate preferences. The differences in substrate specificity can be explained on the basis of the structures of the protein and the substrate. Our results indicate that His259 may be the catalytic base for the reaction, and Glu320, Glu287 bracket the catalytic His259. Especially, Glu320 forms a weak hydrogen bond with His259 and promotes transfer of an H ion.

In situ Localization of Phenylpropanoid-Related Gene Expression in Different Tissues of Light- and Dark-Grown Parsley Seedlings

Using in situ RNA hybridization and immunohistochemical techniques, we investigated the tissue-specific and light-dependent expression of four genes involved in phenylpropanoid metabolism in various parts of parsley (Petroselinum crispum) seedlings. The genes encode phenylalanine ammonia-lyase (PAL) and 4-coum arate: CoA ligase (4CL), two enzymes of general phenylpropanoid metabolism; chalcone synthase (CHS), the key enzyme of flavonoid biosynthesis; and bergaptol O-methyltransferase (BMT), a late enzyme of the furanocoumarin pathway. In young leaves, PAL and 4 CL genes were expressed predominantly and in a coordinated fashion in epidermal cells, young xylem cells of vascular bundles, and epithelial cells of oil ducts. The gene(s) encoding BMT was active exclusively in the epithelial cells of oil ducts, whereas CHS gene expression was largely confined to the epidermis. In shoot and root apices, PAL, 4CL and CHS m RNAs were detectable at low levels without distinct patterns. The corresponding proteins, however, accumulated preferentially in the protoderm and pith meristem of the shoot apex and in root-cap cells. A gene encoding pathogenesis-related protein 1 (PR 1) was analyzed in roots for comparison and was found to be expressed predominantly in the cortical cells of root tips. The expression of all genes investigated was, to a greater or lesser extent, dependent on light conditions and tissue age, with highest levels occurring in newly differentiated, light-exposed tissue. In the leaf epidermis of seedlings illuminated for 20 h, PAL and CHS mRNAs and proteins, as well as 4CL protein, were particularly abundant.

Timing of Changes in Protein Synthesis Pattern in Elicitor-Treated Cell Suspension Cultures of Parsley ( Petroselinum crispum)

Abstract The timing of changes in the protein synthesis pattern of elicitor-treated, [35S]methionine-labelled parsley cells (Petroselinum crispum) was analyzed by two-dimensional gel electropho­ resis. Five groups were distinguished from a large number of elicitor-responsive as well as un­ responsive proteins. Two groups were synthesized de novo either early or late after elicitor ap­ plication; two other groups were strongly reduced in their rates of synthesis either early or late after elicitor application; and one group was not appreciably affected at all. The elicitor-in-duced changes altered the total protein com position considerably. A few selected, induced pro­ teins were functionally identified. These included two early induced enzymes, phenylalanine amm onia-lyase (PAL) and 4-coum arate:C oA ligase (4C L), and a late induced enzyme, a ber-gaptol O-methyltransferase (BM T) which is specifically involved in the biosynthesis of furano-coumarin phytoalexins. The biological significance of the observed differential timing of changes in protein synthesis rates is discussed.

Temporal and Spatial Patterns of Gene Expression around Sites of Attempted Fungal Infection in Parsley Leaves.

We analyzed the expression patterns of several pathogen defense-related genes in primary leaf buds of parsley by in situ RNA hybridization. Labeled antisense RNA probes were generated from seven selected cDNAs detecting transcripts from genes that are rapidly and strongly activated in cultured parsley cells upon treatment with fungal elicitor. These genes encode two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, a furanocoumarin-specific bergaptol O-methyltransferase, one pathogenesis-related protein, and three less well characterized proteins, designated as ELI 3, ELI 5, and ELI 7. In uninfected tissue, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNA levels were high in epidermal cells, oil-duct epithelial cells, and cells of the developing xylem; bergaptol O-methyltransferase mRNA was confined to oil-duct epithelial cells; and the pathogenesis-related protein and ELI 3, ELI 5, and ELI 7 mRNAs were undetectable. All seven mRNAs accumulated transiently and locally around infection sites caused by the soybean-pathogenic fungus Phytophthora megasperma f. sp. glycinea, to which parsley is nonhost-resistant. The observed late appearance of bergaptol O-methyltransferase mRNA, as compared with all other mRNAs, is in accord with a similar relative timing of transient gene activation in elicitor-treated cell cultures. Sharp borders were observed between the infection center, where hypersensitive cell death had occurred in response to fungal penetration, the surrounding area of local gene activation, and the remainder of the tissue not showing any apparent response. Some of the genes were also activated, although less sharply localized, upon wounding of parsley leaves.

Cellular localization of nonhost resistance reactions of parsley (Petroselinum crispum) to fungal infection

The response of parsley seedlings (Petroselinum crispum) inoculated with zoospores of the soybean-pathogenic fungus, Phytophthora megasperma f. sp. glycinea, ranged from "immunity" to "physiological susceptibility" depending on the post-inoculation environmental conditions. Typical nonhost resistance reactions, hypersensitive cell death and the formation of small local lesions, occurred under high relative humidity and 16 h illumination per day. Localized biochemical reactions were monitored using fluorescence microscopy combined with histochemical and immunohistochemical methods. The rapid accumulation of furanocoumarin phytoalexins, wall-bound phenolics and callose was detected around infection sites. Indirect antibody staining of frozen tissue sections demonstrated the local accumulation of phenylalanine ammonia-lyase, a key enzyme of general phenylpropanoid metabolism, and S-adenosyl-L-methionine: bergaptol O-methyltransferase, a specific enzyme of the furanocoumarin pathway. The results indicate that phenylpropanoid derivatives are synthesized de novo at infection sites.

Differential regulation and tissue-specific distribution of enzymes of phenylpropanoid pathways in developing parsley seedlings

Characteristic enzymes of general phenylpropanoid metabolism (phenylalanine ammonialyase) and of the flavonoid-glycoside and furanocoumarin branch pathways (chalcone synthase and S-adenosyl-L-methionine: bergaptol O-methyltransferase, respectively) were localized immuno-histochemically in cross-sections of various aerial parts of parsley (Petroselinum crispum) at different stages of seedling development. Phenylalanine ammonia-lyase occurred predominantly in epidermal and oil-duct epithelial cells, but was also detectable in other tissue parts. The two pathway-specific enzymes were localized in the epidermis (chalcone synthase) and in oil ducts (bergaptol O-methyl-transferase). High chalcone-synthase concentrations occurred very early in leaf development and then declined. High levels of the methyltransferase were present at all times investigated. The temporal and spatial at all times investigated. The temporal and spatial distribution of all three enzymes is in agreement with the time courses and sites of accumulation of the biosynthetic end products.