Functional analysis of the amine substrate specificity domain of pepper tyramine and serotonin N-hydroxycinnamoyltransferases

Pepper (Capsicum annuum) serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N-hydroxycinnamic acid amides of serotonin, including feruloylserotonin and p-coumaroylserotonin. To elucidate the domain or the key amino acid that determines the amine substrate specificity, we isolated a tyramine N-hydroxycinnamoyltransferase (THT) gene from pepper. Purified recombinant THT protein catalyzed the synthesis of N-hydroxycinnamic acid amides of tyramine, including feruloyltyramine and p-coumaroyltyramine, but did not accept serotonin as a substrate. Both the SHT and THT mRNAs were found to be expressed constitutively in all pepper organs. Pepper SHT and THT, which have primary sequences that are 78% identical, were used as models to investigate the structural determinants responsible for their distinct substrate specificities and other enzymatic properties. A series of chimeric genes was constructed by reciprocal exchange of DNA segments between the SHT and THT cDNAs. Functional characterization of the recombinant chimeric proteins revealed that the amino acid residues 129 to 165 of SHT and the corresponding residues 125 to 160 in THT are critical structural determinants for amine substrate specificity. Several amino acids are strongly implicated in the determination of amine substrate specificity, in which glycine-158 is involved in catalysis and amine substrate binding and tyrosine-149 plays a pivotal role in controlling amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of tyrosine is corroborated by the THT-F145Y mutant that uses serotonin as the acyl acceptor. The results from the chimeras and the kinetic measurements will direct the creation of additional novel N-hydroxycinnamoyltransferases from the various N-hydroxycinnamoyltransferases found in nature.     

Biosynthesis and biotechnological production of serotonin derivatives

Serotonin derivatives belong to a class of phenylpropanoid amides found at low levels in a wide range of plant species. Representative serotonin derivatives include feruloylserotonin (FS) and 4-coumaroylserotonin (CS). Since the first identification of serotonin derivatives in safflower seeds, their occurrence, biological significance, and pharmacological properties have been reported. Recently, serotonin N-hydroxycinnamoyl transferase (SHT), which is responsible for the synthesis of serotonin derivatives, was cloned from pepper (Capsicum annuum) and characterized in terms of its enzyme kinetics. Using the SHT gene, many attempts have been made to either increase the level of serotonin derivatives in transgenic plants or produce serotonin derivatives de novo in microbes by dual expression of key genes such as SHT and 4-coumarate-CoA ligase (4CL). Due to the strong antioxidant activity and other therapeutic properties of serotonin derivatives, these compounds may have high potential in treatment and prophylaxis, as cosmetic ingredients, and as major components of functional foods or feeds that have health-improving effects. This review examines the biosynthesis of serotonin derivatives, corresponding enzymes, heterologous production in plants or microbes, and their applications.     

Peroxidases and the metabolism of hydroxycinnamic acid amides in Poaceae

The arsenal of plants to fight off microorganisms and herbivores include hydroxycinnamic acid amides (HCAA) and their oxidation products. Hydroxycinnamic acid amides are widespread in the plant kingdom and in the recent years our knowledge of their biosynthesis and catabolism has increased substantially. Peroxidases are the primary candidates as the oxidative enzymes responsible for the turnover of hydroxycinnamic acid amide monomers. In barley, hydroxycinnamoylagmatine derivatives accumulate in young seedlings and in tissues infected with fungi. Hydroxycinnamoylagmatine is found as anti-fungal soluble dimers, called hordatines, and it is also a likely constituent of cell walls. Current evidence suggest that peroxidases are involved in the cross-linking of hydroxycinnamoylagmatine with cell wall components and possibly also in the synthesis of hordatines. Epidermal cell walls of barley respond to infection by the powdery mildew fungus with the deposition of polyphenolic material, that apparently contains hydroxycinnamic acid amides, at the site of attempted penetration. Accumulation of these compounds lowers the successful penetration by the fungus. The recent characterization of agmatine coumaroyl transferase (ACT), the N-hydroxycinnamoyltransferase responsible for the synthesis of hydroxycinnamoylagmatine in barley, has indicated that the production of these metabolites is widespread in the plant body and suggests multiple physiological functions for HCAA derivatives. The cloning of ACT has enabled the revelation of homologues genes in several monocots and the presence of a range of structurally diverse HCAAs in cereals suggests that their peroxidase-mediated metabolism is a common theme. The prospects for metabolic engineering of these pathways into other crops are discussed. Abbreviations: HCAA – hydroxycinnamic acid amide; HRPC – horseradish peroxidase C; ACT – agmatine coumaroyl transferase; THT – tyramine hydroxycinnamoyl transferase; HCBT – hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyl transferase; PHT – putrescine hydroxycinnamoyl transferase; SHT – spermidine/spermine hydroxycinnamoyl transferase; HHT – hydroxyanthranilate hydroxycinnamoyl transferase; p-CHA –p-coumaroyl hydroxyagmatine; p-CHDA –p-coumaroyl hydroxydehydroagmatine; PAL – phenylalanine ammonia lyase.     

Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase

As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides. Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response. To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation. While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds. The major one was isolated and identified as tyrosol glucoside by liquid chromatography-electrospray ionization-high resolution mass spectrometry and NMR analyses. Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.     

Cloning and characterization of a hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase induced in response to UV-C and wounding from Capsicum annuum

Hydroxycinnamoyl-CoA : tyramine N-(hydroxycinnamoyl) transferase (THT) is a pivotal enzyme in the synthesis of N-(hydroxycinnamoyl)-amines, which are associated with cell wall fortification in plants. The cDNA encoding THT was cloned from the leaves of UV-C treated Capsicum annuum (hot pepper) using a differential screening strategy. The predicted protein encoded by the THT cDNA is 250 amino acids in length and has a relative molecular mass of 28,221. The protein sequence derived from the cDNA shares 76% and 67% identity with the potato and tobacco THT protein sequences, respectively. The recombinant pepper THT enzyme was purified using a bacterial overexpression system. The purified enzyme has a broad substrate specificity including acyl donors such as cinnamoyl-, sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-CoA and acceptors such as tyramine and octopamine. In UV-C treated plants, the THT mRNA was strongly induced in leaves, and the elevated level of expression was stable for up to 36 h. THT mRNA also increased in leaves that were detached from the plant but not treated with UV-C. THT expression was measured in different plant tissues, and was constitutive at a similar level in leaf, root, stem, flower and fruit. Induction of THT mRNA was correlated with an increase in THT protein.     

Characterization of rice tryptophan decarboxylases and their direct involvement in serotonin biosynthesis in transgenic rice

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K _m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.     

Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC xTYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.     

Methanol elicits the biosynthesis of 4-coumaroylserotonin and feruloylserotonin in rice seedlings

Rice (Oryza sativa cv. Dongjin) plants responded to treatment with methanol by inducing the synthesis of secondary metabolites such as serotonin derivatives, which include feruloylserotonin and 4-coumaroylserotonin. This response was not only a dose dependence on methanol showing a maximum effect with 1% methanol concentration, but also methanol specific. No other solvents such as ethanol, atetaldehyde, isopropanol, formaldehyde and formic acid showed the induced synthesis of serotonin derivatives as methanol did. The methanol induced synthesis of serotonin derivatives was completely blocked by the addition of abscisic acid (ABA), and significantly inhibited by the additions of zeatin and indoleacetic acid (IAA). However, gibberellic acid (GA) had little effect on the action of methanol. Finally, the induced synthesis of serotonin derivatives upon methanol treatment was closely associated with the transient increase in the activity of key enzyme of serotonin N-hydroxycinnamoyl transferase (SHT) which catalyzes the condensation of serotonin and phenolic-CoA into serotonin derivatives.Key words: elicitor, methanol, 4-coumaroylserotonin, feruloylserotonin, serotonin N-hydroxycinnamoyl transferase, rice seedlingsElicitor broadly refers to molecules and stimuli that either induce or control gene expression and metabolism.¹ To date, a series of elicitors have been reported and include various cell wall constituents of plant and microbe origins, avirulence gene products from microbes, and a lots of chemical and physical stimuli such as CuSO₄, CuCl₂, ozone and UV light.² Among chemical elicitors, CuSO₄ is well known to elicit the accumulation of sesquiterpene lubimin in fruit cavities of Datura stramonium.³ Aluminum chloride (AlCl₃) induces resveratrol synthesis in grapevine leaves.⁴ It is also reported that Arabidopsis induces camalexin synthesis in response to α-aminobutyric acid.⁵ Recently, it was found that rice leaves upon senescence produced methanol which then triggered the synthesis of tryptophan and serotonin, suggestive of a key role of methanol as an endogenous elicitor for both primary and secondary metabolites.⁶ Here, we further examined the role of methanol in rice leaves as an elicitor on the biosynthesis of serotonin derivatives such as 4-coumaroylserotonin (CS) and feruloylserotonin (FS) which show antifungal activity as well as antioxidant activity.^7,8     

Elevated tyrosine decarboxylase and tyramine hydroxycinnamoyltransferase levels increase wound-induced tyramine-derived hydroxycinnamic acid amide accumulation in transgenic tobacco leaves

Feruloyltyramine (FT) and 4-coumaroyltyramine (4CT) participate in the defense of plants against pathogens through their extracellular peroxidative polymerization, which is thought to reduce cell wall digestibility. Hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TYDC; EC 4.1.1.25) are purported to play key roles in the stress-induced regulation of tyramine-derived hydroxycinnamic acid amide (HCAAT) metabolism. Transgenic tobacco (Nicotiana tabacum cv. Xanthi) was engineered to constitutively express tobacco THT. A T₁ plant over-expressing THT was crossbred with T₁ tobacco expressing opium poppy TYDC2, to produce a T₂ line with elevated THT and TYDC activities compared with wild type plants. The effects of an independent increase in TYDC or THT activity, or a dual increase in both TYDC and THT on the cellular pools of HCAAT pathway intermediates and the accumulation of soluble and cell wall-bound FT and 4CT were examined. Increased TYDC activity resulted in a larger cellular pool of tyramine and lower levels of L-phenylalanine in transgenic leaves. In contrast, elevated THT activity reduced tyramine levels. HCAAT levels were low in healthy leaves, but were induced in response to wounding and accumulated around wound sites. Similarly, endogenous THT and TYDC activities were wound-induced. The rate of wound-induced HCAAT accumulation was highest in transgenic plants with elevated THT and TYDC activities showing that both enzymes exert control over the flux of intermediates involved in HCAAT biosynthesis under some conditions.     

Induced tyramine overproduction in transgenic rice plants expressing a rice tyrosine decarboxylase under the control of methanol-inducible rice tryptophan decarboxylase promoter

Tyramine, one of the various biogenic amines found in plants, is derived from the aromatic L-amino acid tyrosine through the catalytic reaction of tyrosine decarboxylase (TYDC). Tyramine overproduction by constitutive expression of TYDC in rice plants leads to stunted growth, but an increased number of tillers. To regulate tyramine production in rice plants, we expressed TYDC under the control of a methanol-inducible plant tryptophan decarboxylase (TDC) promoter and generated transgenic T(2) homozygous rice plants. The transgenic rice plants showed normal growth phenotypes with slightly increased levels of tyramine in seeds relative to wild type. Upon treatment with 1% methanol, the transgenic rice leaves produced large amounts of tyramine, whereas no increase in tyramine production was observed in wild-type plants. The methanol-induced accumulation of tyramine in the transgenic rice leaves was inversely correlated with the tyrosine level. These data indicate that tyramine production in rice plants can be artificially controlled using the methanol-inducible TDC promoter, suggesting that this promoter could be used to selectively induce the expression of other proteins or metabolites in rice plants.     

L-Tyrosine beta-naphthylamide is a potent competitive inhibitor of tyramine N-(hydroxycinnamoyl)transferase in vitro

L-Tyrosine beta-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) purified from elicited tobacco cell-suspension cultures. The inhibition is competitive, with the inhibitor binding reversibly to the tyramine binding site of the enzyme. Similar results were obtained with THT extracted from elicited potato cell-suspension cultures. Ki values were found to be 0.66 microM for the enzyme from tobacco and 0.3 microM for the enzyme from potato. L-Tyrosine 7-amido-4-methylcoumarin, a fluorogenic substrate for tyrosine aminopeptidases, the structure of which is close to that of L-tyrosine beta-naphthylamide. was also a powerful inhibitor, but slightly less effective with Ki values of 0.72 and 0.42 microM for tobacco and potato THT, respectively. L-Tyrosine beta-naphthylamide was rapidly hydrolysed when fed in vivo to tobacco or potato cell cultures or when incubated in crude enzymic extracts prepared from these cultures. This hydrolysis, which is presumably catalysed by aminopeptidases, precludes the use of L-tyrosine amides as inhibitors of THT in vivo.     

Tyramine accumulation in rice cells caused a dwarf phenotype via reduced cell division

Transgenic rice plants overexpressing a rice tyrosine decarboxylase (TyDC) exhibited a dwarf phenotype with a high level of tyramine accumulation. The height of transgenic rice was reduced on average to 35% of the wild-type height, whereas the number of tillers increased to 190% that of wild type. When judged by cellular distribution of tyramine and tyramine derivatives, the level of tyramine in soluble and insoluble fractions was higher than that of tyramine derivatives such as 4-coumaroyltyramine (CT) in the transgenic rice plants, suggesting that tyramine rather than its derivatives was a causative compound triggering the dwarf phenotype. Microscopic observation revealed that cell size in the transgenic lines was maintained, with a slightly irregular arrangement in the leaf mesophyll cells. When wild-type rice seeds were grown in the presence of tyramine, rice seedlings also showed stunted phenotypes in a dose-dependent manner. When these stunted seedlings were employed to measure the degree of cellular proliferation by bromodeoxyuridine incorporation, only small numbers of cells were found to retain labeled nuclei in shoot tips compared with the untreated control. These results show that the dwarf phenotype associated with tyramine accumulation in transgenic rice plants is attributable to a reduction in cell number rather than cell size. In addition, our dwarf phenotype caused by tyramine was not closely associated with known dwarf genes such as D88.     

Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.)

Genes encoding seven enzymes of the monolignol pathway were independently downregulated in alfalfa (Medicago sativa) using antisense and/or RNA interference. In each case, total flux into lignin was reduced, with the largest effects arising from the downregulation of earlier enzymes in the pathway. The downregulation of l-phenylalanine ammonia-lyase, 4-coumarate 3-hydroxylase, hydroxycinnamoyl CoA quinate/shikimate hydroxycinnamoyl transferase, ferulate 5-hydroxylase or caffeic acid 3-O-methyltransferase resulted in compositional changes in lignin and wall-bound hydroxycinnamic acids consistent with the current models of the monolignol pathway. However, downregulating caffeoyl CoA 3-O-methyltransferase neither reduced syringyl (S) lignin units nor wall-bound ferulate, inconsistent with a role for this enzyme in 3-O-methylation ofS monolignol precursors and hydroxycinnamic acids. Paradoxically, lignin composition differed in plants downregulated in either cinnamate 4-hydroxylase or phenylalanine ammonia-lyase. No changes in the levels of acylated flavonoids were observed in the various transgenic lines. The current model for monolignol and ferulate biosynthesis appears to be an over-simplification, at least in alfalfa, and additional enzymes may be needed for the 3-O-methylation reactions of S lignin and ferulate biosynthesis.     

Beta-1,3-glucooligosaccharide induced activation of four enzymes responsible for N-p-coumaroyloctopamine biosynthesis in potato (Solanum tuberosum cv.) tuber tissue

Potato tuber disks, when treated with laminarin, a beta-1,3-glucooligosaccharide from Laminaria digitata, accumulate a hydroxycinnamoyl amide compound, N-p-coumaroyloctopamine (p-CO). The biosynthesis of p-CO was investigated by feeding experiments, in order to show that the precursors of N-p-coumaroyl and octopamine moieties of p-CO are L-phenylalanine and L-tyrosine, respectively. The treatment of potato tuber tissue with laminarin resulted in elevated activities of four enzymes which are putatively involved in p-CO biosynthesis: phenylalanine ammonia lyase (PAL; EC 4.3.1.5), 4-hydroxycinnamic acid:CoA ligase (4CL; EC 6.2.1.12), hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25). Among these, the response of TyrDC was specific to laminarin treatment, thus indicating that the regulation of TyrDC activity is critical for the accumulation of p-CO in potato tuber tissue.     

Characterization of tryptamine 5-hydroxylase and serotonin synthesis in rice plants

Serotonin is a well-known pineal hormone that in mammals plays a key role in mood. In plants, serotonin is implicated in several physiological roles such as flowering, morphogenesis, and adaptation to environmental changes. However, its biosynthetic enzyme in plants has not been characterized. Therefore, we measured the serotonin content and enzyme activity responsible for serotonin biosynthesis in rice seedlings. Tryptamine 5-hydroxylase (T5H), which converts tryptamine into serotonin, was found as a soluble enzyme that had maximal activity in the roots. The maximal activity of T5H was closely associated with the enriched synthesis of serotonin in roots. Tetrahydropterine-dependent T5H activity was inhibited by tyramine, tryptophan, 5-OH-tryptophan, and octopamine, but remained unaltered by dopamine in vitro. The tissues of rice seedlings grown in the presence of tryptamine exhibited a dose-dependent increase in serotonin in parallel with enhanced T5H enzyme activity. However, no significant increase in serotonin was observed in rice tissues grown in the presence of tryptophan, suggesting that tryptamine is a bottleneck intermediate substrate for serotonin synthesis.