Conversion of 5-hydroxytryptophan into serotonin by tryptophan decarboxylase in plants, Escherichia coli, and yeast

The L-tryptophan decarboxylase (TDC) gene of rice was heterologously expressed in various organisms. Transgenic rice overexpressing TDC showed accumulation of serotonin upon 5-hydroxytryptophan treatment, which was consistent with the in vitro 5-hydroxytryptophan decarboxylase enzyme activity of purified recombinant rice TDC in a pyridoxal phosphate-dependent manner. Recombinant yeast harboring TDC produced serotonin at the expense of the endogenous 5-hydroxytryptophan levels.     

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.     

Purification of DOPA decarboxylase from bovine striatum

Pyridoxal phosphate-dependent DOPA decarboxylase has been purified from bovine striatum to a specific activity of 1.6 U/mg protein. After ammonium sulfate precipitation (30–60%) it was purified by DEAE-Sephacel, Sephacryl S-200, and TSK Phenyl 5 PW chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. The bovine striatal DOPA decarboxylase is a dimer (subunit M_r = 56000 by SDS-PAGE) with a native M_r of 106000 as judged by chromatography on Sephacryl S-200 and by sedimentation analysis. Similar to the DOPA decarboxylase purified from non-CNS tissues, the bovine striatal enzyme requires free sulfhydryl groups for activity, is strongly inhibited by heavy metal ions, and can decarboxylate 5-hydroxytryptophan as well. It should be noted, however, that the final enzyme preparation is enriched in DOPA decarboxylase activity. The distribution of the DOPA decarboxylase and 5-HTP decarboxylase activities also varies among several bovine brain regions. In addition, heat treatment of the enzyme preparation inactivated the two decarboxylation activities at different rates.Abbreviations AADC Aromatic L-amino Acid Decarboxylase - CNS Central Nervous System - DOPA 3,4-dihydroxyphenylalanine - DTT Dithiothreitol, 5-HTP - 5-hydroxytryptophan - Mr relative molecular weight - PLP pyridoxal 5-phosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Part of this paper was presented at the 1987 Annual Pharmacology and Toxicology Conferences held at University of North Dakota School of Medicine, North Dakota, USA Res Commun Psychol Psychiat Behav 12: 227–228, 1987 (Abstr).     

Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.     

Production of indole alkaloids by metabolic engineering of the tryptophan pathway in rice

Tryptophan decarboxylase (TDC) converts tryptophan (Trp) into tryptamine, consequently increasing the metabolic flow of tryptophan derivatives into the production of secondary metabolites such as indole alkaloids. We inserted an expression cassette containing OsTDC, a putative tryptophan decarboxylase gene from rice, into an expression plasmid vector containing OASA1D, the feedback‐resistant anthranilate synthase alpha‐subunit mutant (OASA1D). Overexpression of OASA1D has been reported to significantly increase Trp levels in rice. The co‐expression of OsTDC and OASA1D in rice calli led to almost complete depletion of the Trp pool and a consequent increase in the tryptamine pool. This indicates that TDC inactivity is a contributory factor for the accumulation of Trp in rice transgenics overexpressing OASA1D. Metabolic profiling of the calli expressing OsTDC and OASA1D revealed the accumulation of serotonin and serotonin‐derived indole compounds (potentially pharmacoactive β‐carbolines) that have not been reported from rice. Rice calli overexpressing OASA1D:OASA1D is a novel system for the production of significant amounts of pharmacologically useful indole alkaloids in rice.     

Purification and characterization of L-DOPA decarboxylase from human kidney

L-DOPA decarboxylase has been purified to homogeneity from post mortem removed human kidneys. Homogeneity was examined by polyacrylamide gel electrophoresis (PAGE) analysis both in the presence and absence of SDS. The enzyme has a molecular weight of 100,000 daltons estimated by gel filtration and 50,000 daltons determined after SDS-PAGE. Human L-DOPA decarboxylase therefore is a dimer. Polyclonal antibodies produced against human L-DOPA decarboxylase react with the 50,000 daltons enzyme subunit after immuno-blotting and also precipitates enzyme activity. Activity against L-DOPA is partially inhibited by 5-hydroxytryptophan (5-HTP). The effect of various cations on L-DOPA decarboxylase activity has also been tested.     

Probing the role of tryptophan-derived secondary metabolism in defense responses against Bipolaris oryzae infection in rice leaves by a suicide substrate of tryptophan decarboxylase

Tryptophan-derived secondary metabolites, including serotonin and its hydroxycinnamic acid amides, markedly accumulate in rice leaves in response to pathogen attack. These compounds have been implicated in the physical defense system against pathogen invasion by being deposited in cell walls. Serotonin is biosynthesized from tryptophan via tryptamine, and tryptophan decarboxylase (TDC) catalyzes the first committed reaction. In this study, (S)-α-(fluoromethyl)tryptophan (S-αFMT) was utilized to investigate the effects of the inhibition of TDC on the defense responses of rice leaves. S-αFMT, enantiospecifically synthesized from l-tryptophan, effectively inhibited TDC activity extracted from rice leaves infected by Bipolaris oryzae. The inhibition rate increased dependently on the incubation time, indicating that S-αFMT served as a suicide substrate. Treatment of rice seedlings with S-αFMT suppressed accumulation of serotonin, tryptamine, and hydroxycinnamic acid amides of serotonin in a dose-dependent manner in B. oryzae-inoculated leaves. The lesions formed on seedlings treated with S-αFMT lacked deposition of brown materials, and those leaves were severely damaged in comparison with leaves without S-αFMT treatment. Administrating tryptamine to S-αFMT-treated leaves restored accumulation of tryptophan-derived secondary metabolites as well as deposition of brown material. In addition, tryptamine administration reduced damage caused by fungal infection. Accordingly, the accumulation of tryptophan-derived secondary metabolites was suggested to be part of the effective defense mechanism of rice.     

Tyrosine Decarboxylase and Dopa Decarboxylase in Drosophila virilis Under Normal Conditions and Heat Stress: Genetic and Physiological Aspects

The activity of tyrosine decarboxylase (TDC) and dopa decarboxylase (DDC) was studied in adults of two lines of Drosophila virilis,contrasting in their reaction to stress conditions. Differences were found in the activity of both enzymes between individuals of the examined lines. Genetic analysis of these differences was made. Each of the two enzymes was found to be controlled by a single gene or, possibly, by a block of closely linked genes. The gene responsible for TDC activity is located on one of the autosomes (excluding chromosome II). DDC activity in D. virilisis regulated by a gene located, apparently, on chromosome II. Adults of the line responding to stress by a stress reaction (r-line) were shown to react to a short-term heat stress (38°C, 60 min) by a decrease in TDC activity. TDC activity in flies of the line incapable of the stress reaction (nr-line) did not alter in such conditions. DDC activity of adults of both lines was found to be unchangeable under stress conditions.     

Tryptophan decarboxylase from Catharanthus roseus cell suspension cultures: purification,molecular and kinetic data of the homogenous protein

The purification of tryptophan decarboxylase from Catharanthus roseus (TDC, E.C.:4.1.1.27), to apparent homogeneity, is described. The enzyme represents a soluble protein with a molecular weight of 115 000±3 000, consisting of 2 identical subunits of 54 000±1 000. The pI was estimated to be 5.9 and the K_m for L-tryptophan was found to be 7.5×10^-5 M. Phenylalanine, tyrosine and DOPA were not decarboxylated by tryptophan decarboxylase from Catharanthus cells. Similar to the aromatic amino acid decarboxylase from hog kidney the enzyme does not appear to be obligatorily dependent on exogenously supplied pyridoxal phosphate, as it seems to contain a certain amount of this cofactor. The average percentage of TDC in the cells was found to be 0.002% in the growth medium while the level increased up to 0.03% when indole alkaloid biosynthesis was induced. The role of the protein as a bottleneck enzyme of indole alkaloid biosynthesis is discussed.     

Induction of serotonin biosynthesis is uncoupled from the coordinated induction of tryptophan biosynthesis in pepper fruits (<Emphasis Type="Italic">Capsicum annuum</Emphasis>) upon pathogen infection

It has been suggested that serotonin biosynthesis is regulated by tryptophan decarboxylase (TDC) in plants. To determine if TDC plays a rate-limiting role in serotonin biosynthesis, two TDC genes, PepTDC1 and PepTDC2, were cloned from pepper (Capsicum annuum L.) fruits infected with anthracnose fungus and their expression was then examined in various organs, including fruit that had been treated with the fungus or various chemicals. PepTDC1 expression was highly induced in pepper fruits after treatment with fungus and ethylene, while PepTDC2 was constitutively expressed at low levels in all pepper tissues. Additionally, predominant induction of PepTDC1 mRNA and TDC enzyme activity was detected in the unripe-green fruit, but not in the ripe-red fruit upon pathogen infection. Higher expression of TDC in unripe-green fruit was closely associated with increased levels of tryptamine, serotonin, and serotonin derivatives. However, unlike the enhanced serotonin synthesis, tryptophan levels responded unchanged when challenged with the pathogen in both the unripe-green fruit and the ripe-red fruit. Expression of two key tryptophan biosynthetic genes, anthranilate synthase (ASα) and tryptophan synthase (TSβ), remained unchanged in response to treatment. Also, anthranilate synthase enzyme activity remained steady regardless of pathogen infection. Taken together, these results suggest that the synthesis of serotonin was regulated by the induction of TDC without a simultaneous increase in tryptophan levels in pepper fruits.     

Tryptophan decarboxylase from transformed roots of Catharanthus roseus

Tryptophan decarboxylase (TDC, EC 4.1.1.28) from Catharanthus roseus hairy roots was purified 80-fold. Antibodies against TDC were obtained and they recognized only one protein of 55 kDa in crude extracts from hairy root cultures. Elicitation of transformed root cultures with macerozyme yielded a marked increase in TDC activity, which was accompanied by a similar increase in the amount of immunoreactive TDC protein. These results suggest that the alkaloid accumulation, produced by elicitation, requires the synthesis of new TDC polypeptide in C. roseus root cultures and establishes important differences in the regulatory control of this enzyme in root cultures compared to developing seedlings, where the posttranslational regulation apparently plays a major role.     

Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle

Summary The rhythm in melatonin production in the rat is driven by a circadian rhythm in the pineal N-acetyltransferase (NAT) activity. Rats adapted to an artificial lighting regime of 12 h of light and 12 h of darkness per day were exposed to an 8-h advance of the light-dark regime accomplished by the shortening of one dark period; the effect of melatonin, triazolam and fluoxetine, together with 5-hydroxytryptophan, on the reentrainment of the NAT rhythm was studied.In control rats, the NAT rhythm was abolished during the first 3 cycles following the advance shift. It reappeared during the 4th cycle; however, the phase relationship between the evening rise in activity and the morning decline was still compressed.Melatonin accelerated the NAT rhythm reentrainment. In rats treated chronically with melatonin at the new dark onset, the rhythm had already reappeared during the 3rd cycle, in the middle of the advanced night, and during the 4th cycle, the phase relationship between the evening onset and the morning decline of the NAT activity was the same as before the advance shift. In rats treated chronically with melatonin at the old dark onset or in those treated with melatonin 8 h, 5 h and 2 h after the new dark onset during the 1st, 2nd and 3rd cycle, respectively, following the advance shift, the NAT rhythm reappeared during the 3rd cycle as well but in the last third of the advanced night only.Neither triazolam nor fluoxetine together with 5-hydroxytryptophan administered around the new dark onset facilitated NAT rhythm reentrainment after the 8-h advance of the light-dark cycle.Abbreviations NAT N-acetyltransferase - LD cycle light-dark cycle - CT circadian time - LD xy light dark cycle comprising x h of light and y h of darkness     

Decarboxylation of p-Tyrosine: A Potential Source of p-Tyramine in Mammalian Tissues

The question of the existence of a p-tyrosine decarboxylase pathway for the formation of p-tyramine in mammalian tissues remains unresolved. Development of a sensitive and specific assay for p-tyrosine decarboxylase has permitted demonstration of this activity in rat tissues and human kidney. Tyrosine decarboxylase was purified to electrophoretic homogeneity by pH 5.0 precipitation, ammonium sulfate precipitation, gel filtration, phenyl-Sepharose chromatography, DEAE-Sephacel chromatography, and preparative isoelectric focusing. A specific rabbit antiserum to tyrosine decarboxylase was also obtained. Purified tyrosine decarboxylase possessed a narrow pH dependency with an optimum at 8.0. Benzene and certain other organic solvents dramatically stimulated tyrosine decarboxylase activity of purified enzyme. Purified tyrosine decarboxylase activity also decarboxylated L-DOPA, 5-hydroxytryptophan, 3,4-dihydroxyphenylserine, o-tyrosine, m-tyrosine, phenylalanine, histidine, and tryptophan, which suggested that the purified enzyme was aromatic L-amino acid decarboxylase. This conclusion was supported by a constant ratio of 5-hydroxytryptophan decarboxylase to tyrosine decarboxylase throughout the purification scheme and by parallel immunoprecipitation of decarboxylase activities by the specific antityrosine decarboxylase antisera. Thus, we report that p-tyrosine is decarboxylated by aromatic L-amino acid decarboxylase and that this metabolic transformation may be an important source of p-tyramine in mammalian tissues. In conclusion, neuronal tissues that synthesize catecholamines or serotonin should now be considered capable of synthesizing p-tyramine and other biogenic amines.     

Induction of de-novo synthesis of tryptophan decarboxylase in cell suspensions of Catharanthus roseus

Tryptophan decarboxylase (EC 4.2.1.27) is synthesized de-novo by Catharanthus roseus cells shortly after the cells have been transferred into culture medium in which monoterpenoid indole alkaloids are formed. The enzyme production, monitored by in-vivo labelling with [³⁵S]methionine and immunoprecipitation, precedes the apparent maximal enzyme activity by 10–12 h. From the time course of the descending enzyme activity after induction, a half-life of 21 h for tryptophan decarboxylase in C. roseus cell suspensions is calculated. A comparison of the polyadenylated-RNA preparations from C. roseus cells indicates that mRNA activity for tryptophan decarboxylase is only detected in cells grown in the production medium. The importance of tryptophan decarboxylase induction with respect to the accumulation of th corresponding alkaloids is discussed.Abbreviation TDC tryptophan decarboxylase     

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxlyase results in a dark brown phenotype and stunted growth

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxlyase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxlyase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5′ hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, dark-brown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the dark-brown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.     

Schistosoma mansoni: Decarboxylation of 5-hydroxytryptophan, l-dopa,and l-histidine in adult and larval schistosomes

Homogenates of adult and larval Schistosoma mansoni, decarboxylate 5-hydroxytryptophan (5-HTP) and l-dopa in vitro, but not l-histidine. The enzyme responsible, similar to mammalian aromatic-l-amino acid carboxy-lyase (EC 4.1.1.28), has a K_m of 5.1 × 10^?5M, a V_max of 1.1 nmole/30 min/mg protein, and high activity at pH 7.9. No evidence for a specific l-histidine decarboxylase (EC 4.1.1.22) was found in either stage of the schistosome. In vivo, schistosomules form serotonin from 5-HTP, detectable by thin-layer chromatography, within 3–4 hr after addition of ¹⁴C-labeled 5-HTP to the medium. Addition of ¹⁴C-labeled tryptophan under similar conditions results in no detectable formation of 5-HTP.     

Effects of 20‐hydroxyecdysone and juvenile hormone on octopamine metabolism in females of Drosophila

The effect of exogenous 20‐hydroxyecdysone (20E) and juvenile hormone (JH) on the activities of the tyrosine decarboxylase (TDC), the first enzyme in octopamine (OA) synthesis, has been studied in young females of wild type D. virilis and D. melanogaster under normal and heat stress (38°C) conditions. Flies fed 20E expressed increased TDC activity in both species. JH application decreased TDC activity in both species. A rise in JH and 20E levels did not prevent a TDC response to heat stress, but changed the response intensity. A long‐term increase in JH titre had no effect on the activity of main OA catabolyzing enzyme, arylalkylamine N‐acetyltransferase, in females of both species. A possible mechanism of regulation of OA levels by 20E and JH in Drosophila females is discussed. © 2009 Wiley Periodicals, Inc.     

Stimulation of the Serotonin Autoreceptor Prevents the Calcium-Calmodulin-Dependent Increase of Serotonin Biosynthesis in Rat Raphe Slices

The role of the serotonin (5-hydroxytryptamine) autoreceptor in the regulation of the activity of tryptophan hydroxylase was investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measuring the accumulation of 5-hydroxytryptophan in the presence of inhibition of aromatic L-amino acid decarboxylase using 3-hydroxy-4-bromobenzyloxy-amine by HPLC with fluorescence detection. Serotonin and its agonists N,N-dimethyl-5-methoxytryptamine and 1-(m-chlorophenyl)-piperazine reduced the formation of 5-hydroxytryptophan to 50-60% at 10(-5) M. The effect of serotonin was reversed by 10(-5) M methiothepin, an antagonist of the serotonin autoreceptor. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), dose-dependently reduced the basal formation of 5-hydroxytryptophan to 40-50% at 10(-6) and 10(-4) M, respectively. W-7 also reduced the activated formation by A-23187 or dibutyryl cyclic AMP in a dose-dependent manner. W-7 had no effect on 5-hydroxytryptophan formation reduced by serotonin at 10(-5) M. These results suggest that the role of the serotonin autoreceptor was related to the prevention of the calcium-calmodulin-dependent activation of tryptophan hydroxylase.     

The effect of submergence,ethylene and gibberellin on polyamines and their biosynthetic enzymes in deepwater-rice internodes

Submergence and treatment with ethylene or gibberellic acid (GA₃) stimulates rapid growth in internodes of deepwater rice (Oryza sativa L. cv. Habiganj Aman II). This growth is based on greatly enhanced rate of cell-division activity in the intercalary meristem (IM) and on increased cell elongation. We chose polyamine biosynthesis as a biochemical marker for cell-division activity in the IM of rice stems. Upon submergence of the plant, the activity of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) in the IM increased six- to tenfold within 8 h; thereafter, SAMDC activity declined. Arginine decarboxylase (ADC; EC 4.1.1.19) showed a similar but less pronounced increase in activity. The activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in the IM was not affected by submergence. The levels of putrescine and spermidine also rose in the IM of submerged, whole plants while the concentration of spermine remained low. The increase in SAMDC activity was localized in the IM while the activity of ADC rose both in the node and the IM above it. The node also contained low levels of ODC activity which increased slightly following submergence. Increased activities of polyamine-synthesizing enzymes in the nodal region of submerged plants probably resulted from the promotion of adventitious root formation in the node. Treatment of excised rice-stem sections with ethylene or GA₃ enhanced the activities of SAMDC and ADC in the IM and inhibited the decline in the levels of putrescine and spermidine. We conclude that SAMDC and perhaps also ADC may serve as biochemical markers for the enhancement of cell-division activity in the IM of deepwater rice.Abbreviations ADC arginine decarboxylase - GA gibberellin - IM intercalary meristem - ODC ornithine decarboxylase - SAM S-adenosylmethionine - SAMDC SAM decarboxylase     

Effect of clofibrate on brain mevalonate-5-pyrophosphate decarboxylase

The effect of clofibrate on the activity of the three mevalonate-activating enzymes has been studied for the first time in brain by reactions carried out using [2-¹⁴C] mevalonic acid as substrate and 105,000g supernatants from 14-day-old chick brain. Mevalonate-5-pyrophosphate decarboxylase was clearly inhibited, while mevalonate kinase and mevalonate-5-phosphate kinase were not significantly affected. The effect of clofibrate on decarboxylase activity was progressive with increasing concentrations (1.25–5.00 mM) of the inhibitor. A transient inhibition and a subsequent activation as a function of clofibrate concentration seemed to occur for mevalonate kinase. Direct measurements of decarboxylase activity utilizing [2-¹⁴C] pyrophosphomevalonate as the specific substrate of this enzyme corroborated these results. Kinetic studies showed that clofibrate competes with the substrate ATP.