Crystallographic Snapshots of the Dunathan and Quinonoid Intermediates provide Insights into the Reaction Mechanism of Group II Decarboxylases.

PLP-dependent enzymes catalyze a plethora of chemical reactions affecting diverse physiological functions. Here we report the structural determinants of the reaction mechanism in a Group II PLP-dependent decarboxylase by assigning two early intermediates. The in-crystallo complexes of the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observation of the active site interactions. The structures reveal that a subtle rearrangement of a conserved Arg residue in concert with a water-mediated interaction with the carboxylate of the Dunathan intermediate, appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid. Modeling indicates that the conformational change of a dynamic catalytic loop to a closed form controls a conserved network of hydrogen bond interactions between catalytic residues to protonate the quinonoid. Our results provide a structural framework to elucidate mechanistic roles of residues that govern reaction specificity and catalysis in PLP-dependent decarboxylation.

Crystal structures clarify cofactor binding of plant tyrosine decarboxylase.

Plant tyrosine decarboxylase (TyrDC) is a group II pyridoxal 5'-phosphate (PLP)-dependent decarboxylase that mainly catalyzes the decarboxylation of tyrosine to tyramine. This is biologically important for diverting essential primary metabolites into secondary metabolic pathways. Intensive studies have characterized the effective of PLP-binding and the substrate specificity of mammalian 3,4-dihydroxyphenyl-l-alanine (Dopa) decarboxylases, a member of group II PLP-dependent decarboxylase. However, the characteristics of PLP binding and substrate specificity of plant TyrDCs remain unknown. In this study, we focus on the PLP binding manner, and determined the crystal structures of the apo and PLP binding form of type II TyrDC from Papaver somniferum (PsTyrDCII and PsTyrDCII-PLP). The structures showed that, unlike mammalian Dopa decarboxylase, the binding of PLP does not induce distinct conformational changes of PsTyrDCII regarding the overall structure, but the PLP binding pocket displays conformational changes at Phe124, His203 and Thr262. Combining structural comparation and the obtained biochemical findings, it is demonstrated that PsTyrDCII does not binds PLP tightly. Such characteristics of PLP binding may be required by its catalytic reaction and substrate binding. The activity of TyrDC probably regulated by the concentration of PLP in cells.

Improving Soluble Expression of Tyrosine Decarboxylase from Lactobacillus brevis for Tyramine Synthesis with High Total Turnover Number.

The soluble expression of tyrosine decarboxylase (TDC) in heterologous host is often challenging. Here, acidic condition was found to be favorable for improving the soluble expression of TDC from Lactobacillus brevis in Escherichia coli, while addition of carbohydrates (such as glucose, arabinose, and fructose) was vital for decreasing the insoluble fraction. By simple pH control and addition of glucose, the specific activity of TDC in crude extract was enhanced to 46.3 U mg-1, 3.67-fold of that produced from LB medium. Optimization of the reaction conditions revealed that Tween-80 was effective in improving the tyramine production catalyzed by TDC, especially at high tyrosine loadings. As much as 400 mM tyrosine could be completely converted into tyramine with a substrate to catalyst ratio of 29.0 g g-1 and total turnover number of 23,300. This study provides efficient strategies for the highly soluble expression of TDC and biocatalytic production of tyramine.

Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding.

Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms of Lactobacillus brevis TDC (LbTDC) were determined. The LbTDC displays only 25% sequence identity with the only reported TDC structure. Site-directed mutagenesis of the conformationally flexible sites and catalytic center was performed to investigate the potential catalytic mechanism. It was found that H241 in the active site plays an important role in PLP binding because it has different conformations in the apo and holo structures of LbTDC. After binding to PLP, H241 rotated to the position adjacent to the PLP pyridine ring. Alanine scanning mutagenesis revealed several crucial regions that determine the substrate specificity and catalytic activity. Among the mutants, the S586A variant displayed increased catalytic efficiency and substrate affinity, which is attributed to decreased steric hindrance and increased hydrophobicity, as verified by the saturation mutagenesis at S586. Our results provide structural information about the residues important for the protein engineering of TDC to improve catalytic efficiency in the green manufacturing of tyramine.

Tyrosine decarboxylase from Lactobacillus brevis: soluble expression and characterization.

Tyrosine decarboxylase (TDC, EC 4.1.1.25) is an enzyme that catalyzes the decarboxylation of l-tyrosine to produce tyramine and CO2. In this study, a 1881-bp tdc gene from Lactobacillus brevis was cloned and heterologously expressed in Escherichia coli BL21 (DE3). Glucose was discovered to play an important role in the soluble expression of rLbTDC. After optimization, recombinant TDC (rLbTDC) was achieved in excellent solubility and a yield of 224mg rLbTDC/L broth. The C-terminal His-Tagged rLbTDC was one-step purified with 90% recovery. Based on SDS-PAGE and gel filtration analysis, rLbTDC is a dimer composed of two identical subunits of approximately 70kDa. Using l-tyrosine as substrate, the specific activity of rLbTDC was determined to be 133.5U/mg in the presence of 0.2mM pyridoxal-5'-phosphate at 40°C and pH 5.0. The Km and Vmax values of rLbTDC were 0.59mM and 147.1µmolmin(-1)mg(-1), respectively. In addition to l-tyrosine, rLbTDC also exhibited decarboxylase activity towards l-DOPA. This study has demonstrated, for the first time, the soluble expression of tdc gene from L. brevis in heterologous host.

Tyramine and phenylethylamine biosynthesis by food bacteria.

Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products.

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine.

Neuronal basis of innate olfactory attraction to ethanol in Drosophila.

The decision to move towards a mating partner or a food source is essential for life. The mechanisms underlying these behaviors are not well understood. Here, we investigated the role of octopamine - the invertebrate analogue of noradrenaline - in innate olfactory attraction to ethanol. We confirmed that preference is caused via an olfactory stimulus by dissecting the function of the olfactory co-receptor Orco (formally known as OR83b). Orco function is not required for ethanol recognition per se, however it plays a role in context dependent recognition of ethanol. Odor-evoked ethanol preference requires the function of Tbh (Tyramine ß hydroxalyse), the rate-limiting enzyme of octopamine synthesis. In addition, neuronal activity in a subset of octopaminergic neurons is necessary for olfactory ethanol preference. Notably, a specific neuronal activation pattern of tyraminergic/octopaminergic neurons elicit preference and is therefore sufficient to induce preference. In contrast, dopamine dependent increase in locomotor activity is not sufficient for olfactory ethanol preference. Consistent with the role of noradrenaline in mammalian drug induced rewards, we provide evidence that in adult Drosophila the octopaminergic neurotransmitter functions as a reinforcer and that the molecular dissection of the innate attraction to ethanol uncovers the basic properties of a response selection system.

Characterization of the tyramine-producing pathway in Sporolactobacillus sp. P3J.

A sporulated lactic acid bacterium (LAB) isolated from cider must was shown to harbour the tdc gene encoding tyrosine decarboxylase. The isolate belonged to the Sporolactobacillus genus and may correspond to a novel species. The ability of the tdc-positive strain, Sporolactobacillus sp. strain P3J, to produce tyramine in vitro was demonstrated by using HPLC. A 7535 bp nucleotide sequence harbouring the putative tdc gene was determined. Analysis of the obtained sequence showed that four tyramine production-associated genes [tyrosyl-tRNA synthetase (tyrS), tyrosine decarboxylase (tdc), tyrosine permease (tyrP) and Na(+)/H(+) antiporter (nhaC)] were present and were organized as already described in other tyramine-producing LAB. This operon was surrounded by genes showing the highest identities with mobile elements: a putative phage terminase and a putative transposase (downstream and upstream, respectively), suggesting that the tyramine-forming trait was acquired through horizontal gene transfer. Transcription analyses of the tdc gene cluster suggested that tyrS and nhaC are expressed as monocistronic genes while tdc would be part of a polycistronic mRNA together with tyrP. The presence of tyrosine in the culture medium induced the expression of all genes except for tyrS. A clear correlation was observed between initial tyrosine concentration and tyramine production combined with an increase in the final pH reached by the culture. Finally, cloning and expression of the tyrP gene in Lactococcus lactis demonstrated that its product catalyses the exchange of tyrosine and tyramine.

PCR amplification and cloning of tyrosine decarboxylase involved in synephrine biosynthesis in Citrus.

BACKGROUND: The phenolic amine synephrine is a vascoconstrictor and bronchiectatic agent and holds promise as an aid to weight management and obesity reduction. Synephrine is structurally similar to the active ingredients of several commercial cold remedies. Some Citrus contain high concentrations of synephrine. An enzyme involved in synephrine biosynthesis, tyrosine decarboxylase (TYDC), is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that decarboxylates tyrosine to yield CO(2) and tyramine. We used PCR to screen, clone and sequence this gene from various synephrine producing and nonproducing Citrus species and varieties to determine if DNA sequence of this gene correlated with synephrine presence. RESULTS: PCR amplification and comparison of DNA sequence indicates DNA sequence differences that may cause production of truncated proteins to be produced in some nonsynephrine producing Citrus. CONCLUSION: Synephrine production may be genetically determined in part by the gene for TYDC.

Isolation and genotype-dependent, organ-specific expression analysis of a Rhodiola rosea cDNA encoding tyrosine decarboxylase.

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of salidroside, a pharmacologically valuable compound of roseroot. A 1520bp cDNA was cloned and sequenced, and turned out to contain an ORF of 963bp, which encodes a protein of 320 amino acids. The expression of the gene was studied by real-time PCR from leaves and roots of both high and low salidroside producer genotype of roseroot. The gene expression analysis showed the gene to be expressed in leaves as well as in roots; however, the expression was significantly higher in roots, which coincides with the fact that salidroside accumulates preferentially in the underground parts of the plant. The expression was also higher in the line accumulating high levels of salidroside, compared to the line with lower salidroside content. The difference in the expression intensity suggests a decisive role for this enzyme in the salidroside biosynthesis.

Gene expression and characterization of a stress-induced tyrosine decarboxylase from Arabidopsis thaliana.

Full-length tyrosine decarboxylase cDNA (TyrDC) from Arabidopsis thaliana was identified by rapid amplification of cDNA ends-PCR and isolated by RT-PCR. The TyrDC mRNA was substantially induced by drought stress and wounding, and was considerably decreased by salt stress. By using TyrDC protein fusions with green fluorescent protein, an intracellular localization to the cytoplasm was shown. Recombinant (His)(6)-TyrDC was expressed in Escherichia coli and enzymatically characterized: it exclusively catalyzed the conversion of L-tyrosine to tyramine, exhibited an optimum temperature of 50 degrees C, and an optimum pH at approximately 8.5-9. Recombinant TyrDC protein formed tetramers, as shown by blue native gel electrophoresis.

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.

Purification and characterization of tyrosine decarboxylase of Lactobacillus brevis IOEB 9809 isolated from wine.

Tyrosine decarboxylase (EC 4.1.1.25) (TDC) from the wine Lactobacillus brevis IOEB 9809 was purified by a rapid procedure involving anion exchange chromatography, ultrafiltration and hydrophobic interaction chromatography. The protein comprised two subunits of identical molecular mass (approximately 70000 Da). Enzyme activity was dependent on exogenously supplied pyridoxal 5'-phosphate and the enzyme was stable at 4 degrees C in the presence of the coenzyme. Optimum pH for the pure enzyme was 5.0. At this pH, TDC exhibited Michaelis-Menten kinetics (K(m) 0.63 mM, V(max) 998 units) and was highly substrate-specific for L-tyrosine. Other amino acids and L-DOPA are not converted by the protein. Tyramine acted as a mixed non-competitive inhibitor. Significant similarities in some biochemical properties were observed with the corresponding decarboxylase enzyme of Streptococcus faecalis, the sole bacterial TDC described to date.

Multiple evolutionary origin of pyridoxal-5'-phosphate-dependent amino acid decarboxylases.

Comparison of the amino acid sequences of nine different pyridoxal-5'-phosphate-dependent amino acid decarboxylases indicated that they can be subdivided into four different groups that seem to be evolutionarily unrelated to each other. Group I is represented by glycine decarboxylase, a component of a multienzyme system; group II comprises glutamate, histidine, tyrosine, and aromatic-L-amino-acid decarboxylases; group III, procaryotic ornithine and lysine decarboxylase as well as the procaryotic biodegradative type of arginine decarboxylase; group IV, eucaryotic ornithine and arginine decarboxylase as well as the procaryotic biosynthetic type of arginine decarboxylase and diaminopimelate decarboxylase. (N-1) profile analysis, a more stringent application of profile analysis, established the homology among the enzymes of each group. A search with the profile of group II indicated a distant relationship with aminotransferases and thus with the alpha family of pyridoxal-5'-phosphate-dependent enzymes. No evidence was obtained that groups I, III and IV were related with other pyridoxal-5'-phosphate-dependent enzymes or any other protein in the database. Unlike the aminotransferases, which, with few possible exceptions, constitute a single group of homologous proteins, the amino acid decarboxylases, by the criterion of profile analysis, have evolved along multiple lineages, in some cases even if they have the same substrate specificity.

A pathogen-responsive gene of parsley encodes tyrosine decarboxylase.

A group of recently isolated parsley (Petroselinum crispum) cDNAs representing genes that are transcriptionally activated upon fungal infection or elicitor treatment have been demonstrated to encode tyrosine decarboxylase (TyrDC). The deduced TyrDC protein sequence shares extensive similarity with two functionally related enzymes, tryptophan decarboxylase from periwinkle and dopa decarboxylase from Drosophila melanogaster. Expression of TyrDC cDNA in Escherichia coli yielded catalytically active protein with high substrate specificity for tyrosine. All four identified parsley TyrDC genes have been cloned and encode at least three TyrDC isozymes. Treatment of cultured parsley cells with fungal elicitor caused very rapid and transient increases in TyrDC mRNA and enzyme activity levels.