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Expression of tryptophan decarboxylase and tyrosine decarboxylase genes in tobacco results in altered biochemical and physiological phenotypes.

The substrate specificity of tryptophan (Trp) decarboxylase (TDC) for Trp and tyrosine (Tyr) decarboxylase (TYDC) for Tyr was used to modify the in vivo pools of these amino acids in transgenic tobacco. Expression of TDC and TYDC was shown to deplete the levels of Trp and Tyr, respectively, during seedling development. The creation of artificial metabolic sinks for Trp and Tyr also drastically affected the levels of phenylalanine, as well as those of the non-aromatic amino acids methionine, valine, and leucine. Transgenic seedlings also displayed a root-curling phenotype that directly correlated with the depletion of the Trp pool. Non-transformed control seedlings could be induced to display this phenotype after treatment with inhibitors of auxin translocation such as 2,3,5-triiodobenzoic acid or N-1-naphthylphthalamic acid. The depletion of aromatic amino acids was also correlated with increases in the activities of the shikimate and phenylpropanoid pathways in older, light-treated transgenic seedlings expressing TDC, TYDC, or both. These results provide in vivo confirmation that aromatic amino acids exert regulatory feedback control over carbon flux through the shikimate pathway, as well as affecting pathways outside of aromatic amino acid biosynthesis.

3-Deoxy-7-Phosphoheptulonate Synthase↗

Purification and characterisation of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase.

Microbial tyrosine decarboxylase (EC 4.1.1.25) and mammalian aromatic-L-amino-acid decarboxylase (EC 4.1.1.28) catalyse the formation of tyramine from L-tyrosine. These enzymes were characterised after isolation to purity by methods including fast polymer liquid chromatography (FPLC). Tyrosine decarboxylase was isolated from Streptococcus faecalis by FPLC anion exchange chromatography (11-times purification; 72% recovery; 23.2 U/mg protein). FPLC on Phenyl-Superose resulted in purification to 115 U/mg protein. Aromatic-L-amino-acid decarboxylase was isolated from pig kidney by ammonium sulfate fractionation, DEAE chromatography, and FPLC anion exchange chromatography (21-times purification; 22% recovery; 0.71 U/mg protein). By FPLC chromatofocusing, tyrosine decarboxylase eluted at pH 4.3 and aromatic-L-amino-acid decarboxylase at pH 5.0. Isoelectric focusing of tyrosine decarboxylase gave two bands (pI 4.4 and 4.5). With pyridoxal 5'-phosphate removed by ultrafiltration, only one band (pI 4.4) appeared, and SDS polyacrylamide electrophoresis confirmed the purity. FPLC gel filtration resulted in molecular weights 143,000 and 86,000, respectively, for tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. In SDS electrophoresis, tyrosine decarboxylase had the monomer molecular weight 75,000, showing a dimer structure for the enzyme.

Chromatography, Gel↗

[Genetic control of Drosophila virilis tyrosine decarboxylase activity].

Tyrosine decarboxylase (TDC) activity was studied in flies of two Drosophila virilis lines having contrasting response to stress. Lines 101 and 147 exhibited different TDC activity. Genetic analysis of these differences was performed. TDC activity was shown to be controlled by one gene or a block of tightly linked genes. The gene controlling TDC activity was mapped to one autosome but not to chromosome 2. In line 101, short-term heat stress (38 degrees C, 60 min) resulted in reduction in TDC activity, whereas, in line 147, this activity remained unchanged.

Animals↗

Some aspects on L-dopa decarboxylase and p-tyrosine decarboxylase in the central nervous and peripheral tissues of the American cockroach Periplaneta americana.

1. Aromatic amino acid decarboxylase activities toward L-DOPA (L-3,4-dihydroxyphenylalanine), 5-HTP (5-hydroxytryptophan) and p-tyrosine in different tissues of the sclerotized and newly ecdysed cockroach were analyzed. 2. The ratios of enzyme activity with regard to L-DOPA and p-tyrosine varied considerably in the tissues and between the two different growth stages. 3. A DOPA decarboxylase and a p-tyrosine decarboxylase were separated by gel filtration and ion exchange chromatography. 4. The optimal pH requirement for both enzymes was 7.5 with the exception of the one decarboxylating 5-HTP. 5. The molecular weights of the cockroach brain DOPA decarboxylase and tyrosine decarboxylase were estimated to be 120,000 and 100,000, respectively. 6. Unlike the mammalian aromatic amino acid decarboxylase, the cockroach DOPA decarboxylase cannot be activated by a small amount of benzene. 7. An increase of over 50-fold of DOPA decarboxylase activity and a 50% reduction of tyrosine decarboxylase activity in the epidermal tissue of the newly ecdysed animals was observed. 8. In the fully sclerotized cockroach, a reversible endogenous inhibitor(s) of DOPA decarboxylase in the integument was observed, suggesting that the DOPA decarboxylase is suppressed in the epidermal tissues when ecdysis does not occur.

Animals↗

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.

Amides↗

Identification of the Enterococcus faecalis tyrosine decarboxylase operon involved in tyramine production.

Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 microg ml(-1) for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes.

Amino Acid Sequence↗

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB 9809: characterization and conservation in tyramine-producing bacteria.

Bacterial genes of tyrosine decarboxylases were recently identified. Here we continued the sequencing of the tyrosine decarboxylase locus of Lactobacillus brevis IOEB 9809 and determined a total of 7979 bp. The sequence contained four complete genes encoding a tyrosyl-tRNA synthetase, the tyrosine decarboxylase, a probable tyrosine permease and a Na+/H+ antiporter. Rapid amplification of cDNA ends (RACE) was employed to determine the 5'-end of mRNAs containing the tyrosine decarboxylase gene. It was located only 34-35 nucleotides upstream of the start codon, suggesting that the preceding tyrosyl-tRNA synthetase gene was transcribed separately. In contrast, reverse transcription-polymerase chain reactions (RT-PCRs) carried out with primers designed to amplify regions spanning gene junctions showed that some mRNAs contained the four genes. Homology searches revealed similar clusters of four genes in the genome sequences of Enterococcus faecalis and Enterococcus faecium. Phylogenetic analyses supported the hypothesis that these genes evolved all together. These data suggest that bacterial tyrosine decarboxylases are encoded in an operon containing four genes.

Amino Acid Sequence↗

Elicitor-induced tyrosine decarboxylase in berberine-synthesizing suspension cultures of Thalictrum rugosum.

Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline-derived alkaloids.

Berberine↗

Decreased cell wall digestibility in canola transformed with chimeric tyrosine decarboxylase genes from opium poppy

Tyrosine decarboxylase (TYDC) is a common plant enzyme involved in the biosynthesis of numerous secondary metabolites, including hydroxycinnamic acid amides. Although a definite function has not yet been determined, amides have been proposed to form a physical barrier against pathogens because they are usually found as integral cell wall components. Canola (Brassica napus) was independently transformed with chimeric genes (35S::TYDC1 and 35S::TYDC2) under the transcriptional control of the cauliflower mosaic virus 35S promoter, and encoding two TYDC isoforms from opium poppy (Papaver somniferum). All T0 plants displayed a suppressed level of wild-type TYDC activity, and transgene mRNAs were not detected. Silencing of 35S::TYDC1 was overcome in the T1 progeny of self-pollinated T0 plants, since high levels of TYDC1 mRNAs were detected, and TYDC activity increased up to 4-fold compared with wild-type levels. However, TYDC1 mRNA levels decreased in T2 plants and were not detected in the T3 progeny. TYDC activity also gradually declined in T2 and T3 plants to nearly wild-type levels. In contrast, silencing of 35S::TYDC2 was maintained through four consecutive generations. T1 plants with a 3- to 4-fold increase in wild-type TYDC activity showed a 30% decrease in cellular tyrosine pools and a 2-fold increase in cell wall-bound tyramine compared with wild-type plants. An increase in cell wall-bound aromatic compounds was also detected in these T1 plants by ultraviolet autofluorescence microscopy. The relative digestibility of cell walls measured by protoplast release efficiency was inversely related to the level of TYDC activity.

Journal Article↗

Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : II. Partial Characterization.

Properties of purified l-tyrosine decarboxylase (EC 4.1.1.25) from elicitor-induced cell suspension cultures of Eschscholtzia californica Cham. and Thalictrum rugosum Ait. are described. l-Tyrosine decarboxylase is a dimeric enzyme with a molecular weight of 112,600 +/- 600 daltons. The isoelectric point was estimated to be at pH 5.2 and pH 5.4 for the enzyme from E. californica and T. rugosum, respectively. The purified enzymes were stabilized in the presence of pyridoxal-5-phosphate. Optimum pH for the enzyme from both plants was found to be 8.4. Enzyme activity was dependent on exogeneously supplied pyridoxal-5-phosphate. The enzyme decarboxylated l-tyrosine and l-beta-3,4-dihydroxyphenylalanine but was inactive toward l-phenylalanine and l-tryptophan. Apparent K(m) values of Eschscholtzia- and Thalictrum-decarboxylase for l-tyrosine were 0.25 +/- 0.03 and 0.27 +/- 0.04 millimolar, respectively. Similar affinities were found for l-3,4-dihydroxyphenylalanine. Eschscholtzial-tyrosine decarboxylase was strongly inhibited by the phenylalanine analogue l-alpha-aminooxy-beta-phenylpropionate and largely unaffected by d,l-alpha-monofluoromethyl-3,4-dihydroxyphenylalanine and alpha-difluoromethyltyrosine.

Journal Article↗

The steady state kinetics of tyrosine decarboxylase from Streptococcus faecalis.

The present study has explained the general reaction mechanism of the bacterial tyrosine decarboxylase. The rate equation for this mechanism has been presented. The steady state kinetics of tyrosine decarboxylase, as for tyrosine transaminase, have shown that the apoenzyme can bind not only the coenzyme, but also the non-enzymatically formed Schiff base between the coenzyme and the substrate. Our data then have confirmed the importance of the non-enzymatically formed Schiff base in the B6-dependent enzymes, possibly in all of them which have a low affinity constant for the coenzyme, such that the coenzyme must be present in excess in respect to the protein to saturate the active center. The interaction between apotyrosine decarboxylase with pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate has been studied.

Enterococcus faecalis↗

Sequencing of the tyrosine decarboxylase cluster of Lactococcus lactis IPLA 655 and the development of a PCR method for detecting tyrosine decarboxylating lactic acid bacteria.

The enzymatic decarboxylation of tyrosine produces tyramine, the most abundant biogenic amine in dairy products-especially in cheeses. The screening of lactic acid bacteria isolated from different artisanal cheeses and a number of microbial collections identified 22 tyramine-producing strains belonging to different genera. The Lactococcus lactis strain IPLA 655 was selected, and the genes encoding a putative tyrosyl tRNA synthetase, a tyrosine decarboxylase (tdcA), and a tyrosine-tyramine antiporter, found together as a cluster, were sequenced. The disruption of tdcA yielded a strain unable to produce tyramine. Comparison of the L. lactis IPLA 655 tdcA gene with database tdcA sequences led to the design of two primers for use in a PCR method that identified potential tyramine-producing strains. The proposed method can use purified DNA, isolated colonies, milk, curd, and even cheese as a template. Molecular tools for the rapid detection of tyramine-producing bacteria at any time during the fermentation process could help prevent tyramine accumulation in fermented foods. The proposed technique could be of great use to the food industry.

Amino Acid Sequence↗

Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : I. Induction and Purification.

l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO(2) produced per minute per milligram protein at pH 8.4 and 30 degrees C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 +/- 300 daltons.

Journal Article↗

Tyrosine decarboxylase. Spectrophotometric assay and application in determining pyridoxal-5'-phosphate.

We have developed a highly sensitive and rapid spectrophotometric assay for tyrosine decarboxylase that can be applied to determining pyridoxal-5'-phosphate. In the assay, tyramine, a product of tyrosine decarboxylation, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a product soluble in toluene whereas tyrosine does not. We determined the amount of tyramine produced enzymatically by reading the absorbance at 340 nm of a toluene extract of the reaction mixture. This method is capable of detecting as low as 2.9 micrograms/mL of the enzyme. Using this method, we find the Km for tyrosine decarboxylase from Streptococcus faecalis to be 3.55 X 10(-4)M. We have also developed a specific and extremely sensitive method for determining pyridoxal-5'-phosphate, a cofactor of the enzyme, by using this spectrophotometric assay with apotyrosine decarboxylase.

Enterococcus faecalis↗

Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB 9809.

Some lactic acid bacteria contain a tyrosine decarboxylase (TDC) which converts tyrosine to tyramine, a biogenic amine frequently encountered in fermented food and wine. Purification and microsequencing of the TDC of Lactobacillus brevis IOEB 9809 allowed us to determine a partial sequence of the TDC gene encoding 264 amino acids of the enzyme. Analysis of this protein sequence revealed typical features of pyridoxal phosphate-dependent amino acid decarboxylases while not any known decarboxylase was closely related to the TDC of L. brevis IOEB 9809. In addition, we could detect other L. brevis strains carrying a TDC gene in a rapid assay based on the polymerase chain reaction.

Amino Acid Sequence↗

Stereospecificity of sodium borohydride reduction of tyrosine decarboxylase from Streptococcus faecalis.

Sodium boro[3H]hydride reduction of tyrosine decarboxylase from Streptococcus faecalis followed by complete hydrolysis of the enzyme produces epsilon-[3H]pyridoxyllysine. Degradation of this material to [4'-3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase shows that the re side at C-4' of the cofactor is exposed to solvent at pH 5.5 and 7.0. After binding of L-tyrosine at pH 5.5 or tyramine at pH 7.0 to the holoenzyme, sodium boro[3H]hydride reduction proceeds from the si face at C-4' of the substrate . cofactor complex. This indicates one of two conformational changes occurs upon binding of substrate; either rotation about the C-4 to C-4' bond in the cofactor or rotation about the axis through the C-5 and C-5' bond.

Binding Sites↗