Functional characterization of phenylalanine ammonia-lyase- and cinnamate 4-hydroxylase-encoding genes from Lycoris radiata, a galanthamine-producing plant.

Galanthamine (GAL), the well-known Amaryllidaceae alkaloid, is a clinically used drug for the treatment of Alzheimer's disease. L-Phenylalanine (Phe) and trans-cinnamic acid (CA) were enzymatically transformed into the catechol portion of GAL. Herein, a Phe ammonia-lyase-encoding gene LrPAL3 and a cinnamate 4-hydroxylase-encoding gene LrC4H were cloned from Lycoris radiata, a GAL-producing plant. LrPAL3 was overexpressed in Escherichia coli and purified to homogeneity. LrPAL3 catalyzes the forward deamination conversion of L-Phe into trans-CA. The 3-chloro- and 4-fluoro-L-Phe were deaminated to generate the corresponding 3-chloro- and 4-fluoro-trans-CA by LrPAL3. LrPAL3-catalyzed reverse hydroamination was confirmed by the conversion of trans-CA into L-Phe with exceptional regio- and stereo-selectivity. LrC4H was overexpressed in E. coli with tCamCPR, a cytochrome P450 reductase-encoding gene. LrC4H catalyzes the regioselective para-hydroxylation on trans-CA to form p-coumaric acid. The transcriptional levels of both LrPAL3 and LrC4H were positively associated with the GAL contents within the leaves and flowers of L. radiata, which suggested that their expression and function are co-regulated and involved in the biosynthesis of GAL. The present investigations on the biosynthetic genes of GAL will promote the development of synthetic biology platforms for this kind of important drug via metabolic engineering.

[Cloning and expression analysis of cinnamate 4-hydroxylase (C4H) reductase gene from Aquilaria sinensis].

The study aimed to clone the open reading frame of cinnamate 4-hydroxylase (C4H) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. One unique sequence containing C4H domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of C4H was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. C4H expression profiles in responds to MeJA (methyl jasmonate) application were analyzed by real-time PCR. The length of C4H open reading frame (ORF) was 1 515 bp, encoding 514 amino acids. The GenBank accession number is KF134783. Inducible-experiments showed that the genes were induced by mechanical wound as well as MeJA induction, and reached the highest expression level at 8 h and 20 h, respectively. The full-length cDNA of C4H and its expression patterns will provide a foundation for further research on its function in the molecular mechanisms of aromatic compounds and flavonoids biosynthesis.

Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in Carthamus tinctorius.

Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3-6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6-24 h and 3-6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.

Substrates and enzyme activities related to biotransformation of resveratrol from phenylalanine by Alternaria sp. MG1.

To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO4, and 0.2 g/L CaSO4 and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 µg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously.

Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis.

The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.

Molecular cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase in the phenylpropanoid biosynthesis pathway in garlic (Allium sativum).

The cDNAs encoding phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) were cloned from garlic (Allium sativum) using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE) PCR. Amino acid sequence alignments showed that AsPAL and AsC4H have more than 70% amino acid identity with their homologues in other plants. The expression of AsPAL and AsC4H transcripts was highest in the roots but surprisingly low in the bulbils, where phenylpropanoid compounds are most concentrated. These results suggest that some phenylpropanoids are synthesized in the roots and subsequently transported to the bulbils of A. sativum .

Molecular cloning and characterization of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and genes involved in flavone biosynthesis in Scutellaria baicalensis.

The involvement of genes in flavones biosynthesis was investigated in different organs and suspension cells obtained from Scutellaria baicalensis. Three full-length cDNAs encoding phenylalanine ammonia-lyase isoforms (SbPAL1, SbPAL2, and SbAPL3) and one gene encoding cinnamate 4-hydroxylase (SbC4H) from S. baicalensis were isolated using rapid amplification of cDNA ends (RACE)-PCR. These cDNAs were used together with previously-isolated clones for 4-coumaroyl CoA ligase (4CL) and chalcone synthase (CHS) to show the expression level in different organs of S. baicalensis. These genes were upregulated in suspension cells of S. baicalensis with biotic/abiotic stress factors. The baicalin and baicalein contents in roots were 22 and 107 times higher than those in flowers, respectively. The treatment of suspension cells with methyl jasmonate (MeJa) enhanced the major flavones in S. baicalensis. Cumulatively, the results of this study should advance ability to biosynthesize important and useful medicinal compounds from a variety of plant species.

Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis.

The initial reactions of the phenylpropanoid pathway convert phenylalanine to p-coumaroyl CoA, a branch point metabolite from which many phenylpropanoids are made. Although the second enzyme of this pathway, cinnamic acid 4-hydroxylase (C4H), is well characterized, a mutant for the gene encoding this enzyme has not yet, to our knowledge, been identified, presumably because knock-out mutations in this gene would have severe phenotypes. This work describes the characterization of an allelic series of Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, each of which harbor mis-sense mutations in C4H (At2g30490). Heterologous expression of the mutant proteins in Escherichia coli yields enzymes that exhibit P420 spectra, indicative of mis-folded proteins, or have limited ability to bind substrate, indicating that the mutations we have identified affect protein stability and/or enzyme function. In agreement with the early position of C4H in phenylpropanoid metabolism, ref3 mutant plants accumulate decreased levels of several different classes of phenylpropanoid end-products, and exhibit reduced lignin deposition and altered lignin monomer content. Furthermore, these plants accumulate a novel hydroxycinnamic ester, cinnamoylmalate, which is not found in the wild type. The decreased C4H activity in ref3 also causes pleiotropic phenotypes, including dwarfism, male sterility and the development of swellings at branch junctions. Together, these observations indicate that C4H function is critical to the normal biochemistry and development of Arabidopsis.

Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus).

Cinnamate 4-hydroxylase (C4H) is a key enzyme of phenylpropanoid pathway, which synthesizes numerous secondary metabolites to participate in development and adaption. Two C4H isoforms, the 2192-bp BnC4H-1 and 2108-bp BnC4H-2, were cloned from oilseed rape (Brassica napus). They both have two introns and a 1518-bp open reading frame encoding a 505-amino-acid polypeptide. BnC4H-1 is 57.73 kDa with an isoelectric point of 9.11, while 57.75 kDa and 9.13 for BnC4H-2. They share only 80.6% identities on nucleotide level but 96.6% identities and 98.4% positives on protein level. Showing highest homologies to Arabidopsis thaliana C4H, they possess a conserved p450 domain and all P450-featured motifs, and are identical to typical C4Hs at substrate-recognition sites and active site residues. They are most probably associated with endoplasmic reticulum by one or both of the N- and C-terminal transmembrane helices. Phosphorylation may be a necessary post-translational modification. Their secondary structures are dominated by alpha helices and random coils. Most helices locate in the central region, while extended strands mainly distribute before and after this region. Southern blot indicated about 9 or more C4H paralogs in B. napus. In hypocotyl, cotyledon, stem, flower, bud, young- and middle-stage seed, they are co-dominantly expressed. In root and old seed, BnC4H-2 is dominant over BnC4H-1, with a reverse trend in leaf and pericarp. Paralogous C4H numbers in Brassicaceae genomes and possible roles of conserved motifs in 5' UTR and the 2nd intron are discussed.