RESUMEN

Aromatic amino acid decarboxylases (AAADs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that catalyze the decarboxylation of aromatic amino acid l-amino acids. In plants, apart from canonical AAADs that catalyze the straightforward decarboxylation reaction, other members of the AAAD family function as aromatic acetaldehyde synthases (AASs) and catalyze more complex decarboxylation-dependent oxidative deamination. The interconversion between a canonical AAAD and an AAS can be achieved by a single tyrosine-phenylalanine mutation in the large catalytic loop of the enzymes. In this work, we report implicit ligand sampling (ILS) calculations of the canonical l-tyrosine decarboxylase from Papaver somniferum (PsTyDC) that catalyzes l-tyrosine decarboxylation and its Y350F mutant that instead catalyzes the decarboxylation-dependent oxidative deamination of the same substrate. Through comparative analysis of the resulting three-dimensional (3D) O2 free energy profiles, we evaluate the impact of the key tyrosine/phenylalanine mutation on oxygen accessibility to both the wild type and Y350F mutant of PsTyDC. Additionally, using molecular dynamics (MD) simulations of the l-tryptophan decarboxylase from Catharanthus roseus (CrTDC), we further investigate the dynamics of a large catalytic loop known to be indispensable to all AAADs. Results of our ILS and MD calculations shed new light on how key structural elements and loop conformational dynamics underlie the enzymatic functions of different members of the plant AAAD family.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático , Dominio Catalítico , Simulación de Dinámica Molecular , Oxígeno , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/química , Oxígeno/metabolismo , Oxígeno/química , Papaver/enzimología , Papaver/genética , Papaver/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Tirosina/metabolismo , Tirosina/química , Tirosina/genética

RESUMEN

Aromatic L-amino acid decarboxylases (AADCs) catalyze the conversion of aromatic L-amino acids into aromatic monoamines that play diverse physiological and biosynthetic roles in living organisms. For example, dopamine and serotonin serve as major neurotransmitters in animals, whereas tryptamine and tyramine are essential building blocks for synthesizing a myriad of secondary metabolites in plants. In contrast to the vital biological roles of AADCs in higher organisms, microbial AADCs are found in rather a limited range of microorganisms. For example, lactic acid bacteria are known to employ AADCs to achieve intracellular pH homeostasis and engender accumulation of tyramine, causing a toxic effect in fermented foods. Owing to the crucial pharmaceutical implications of aromatic monoamines and their derivatives, synthetic applications of AADCs have attracted growing attention. Besides, recent studies have uncovered that AADCs of human gut microbes influence host physiology and are involved in drug availability of Parkinson's disease medication. These findings bring the bacterial AADCs into a new arena of extensive research for biomedical applications. Here, we review catalytic features of AADCs and present microbial applications and challenges for biotechnological exploitation of AADCs. KEY POINTS: • Aromatic monoamines and their derivatives are increasingly important in the drug industry. • Aromatic L-amino acid decarboxylases are the only enzyme for synthesizing aromatic monoamines. • Microbial applications of aromatic L-amino acid decarboxylases have drawn growing attention.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático , Carboxiliasas , Aminoácidos Aromáticos , Animales , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Serotonina/metabolismo , Tiramina/metabolismo

RESUMEN

Aromatic amino acid decarboxylase (AADC) deficiency is a rare, autosomal recessive neurometabolic disorder caused by mutations in the DDC gene, leading to a deficit of AADC, a pyridoxal 5'-phosphate requiring enzyme that catalyzes the decarboxylation of L-Dopa and L-5-hydroxytryptophan in dopamine and serotonin, respectively. Although clinical and genetic studies have given the major contribution to the diagnosis and therapy of AADC deficiency, biochemical investigations have also helped the comprehension of this disorder at a molecular level. Here, we reported the steps leading to the elucidation of the functional and structural features of the enzyme that were useful to identify the different molecular defects caused by the mutations, either in homozygosis or in heterozygosis, associated with AADC deficiency. By revisiting the biochemical data available on the characterization of the pathogenic variants in the purified recombinant form, and interpreting them on the basis of the structure-function relationship of AADC, it was possible: (i) to define the enzymatic phenotype of patients harboring pathogenic mutations and at the same time to propose specific therapeutic managements, and (ii) to identify residues and/or regions of the enzyme relevant for catalysis and/or folding of AADC.

Asunto(s)

Errores Innatos del Metabolismo de los Aminoácidos/etiología , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/deficiencia , Susceptibilidad a Enfermedades , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Biomarcadores , Catálisis , Dopamina/metabolismo , Homocigoto , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Serotonina/metabolismo , Relación Estructura-Actividad

RESUMEN

Tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyzes tryptophan decarboxylation to form tryptamine through the cofactor pyridoxal-5'-phosphate (PLP), a crucial stage in the production of the terpenoid indole alkaloids like camptothecin (CPT). A new gene encoding TDC was identified from the CPT-producing plant Ophiorrhiza pumila by transcriptome analysis, termed OpTDC2. It contained a 1,536 bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01 kDa and an isoelectric point of 6.39. Multiple sequence alignment and phylogenetic tree analysis showed the closest similarity (85%) with the TDC from Mitragyna speciosa. Moreover, the highest expression of OpTDC2 was observed in the O. pumila root. To achieve high-efficiency expression of OpTDC2 in Escherichia coli, we fused the TF tag onto the N-terminal of the OpTDC2. Optimum enzymatic activity was observed at 45 °C, pH 8 and cofactor concentration of 0.1 mM. The catalytic reaction was strongly inhibited by metal ions of Cu2+ , Zn2+ , and Fe2+ . The l-tryptophan was particularly catalyzed compared with d-tryptophan. Besides, the Km and kcat of the OpTDC2 were 1.08 mM and 0.78 Sec-1 , respectively. The results provided information on new functional OpTDC2 that might be used in synthetic biology for the enhanced biosynthesis of CPT in O. pumila.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático , Clonación Molecular , Filogenia , Proteínas de Plantas , Rubiaceae , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Rubiaceae/enzimología , Rubiaceae/genética

RESUMEN

KEY MESSAGE: WsWRKY1-mediated transcriptional modulation of Withania somnifera tryptophan decarboxylase gene (WsTDC) helps to regulate fruit-specific tryptamine generation for production of withanamides. Withania somnifera is a highly valued medicinal plant. Recent demonstration of novel indolyl metabolites called withanamides in its fruits (berries) prompted us to investigate its tryptophan decarboxylase (TDC), as tryptophan is invariably a precursor for indole moiety. TDC catalyzes conversion of tryptophan into tryptamine, and the catalytic reaction constitutes a committed metabolic step for synthesis of an array of indolyl metabolites. The TDC gene (WsTDC) was cloned from berries of the plant and expressed in E. coli. The recombinant enzyme was purified and characterized for its catalytic attributes. Catalytic and structural aspects of the enzyme indicated its regulatory/rate-limiting significance in generation of the indolyl metabolites. Novel tissue-wise and developmentally differential abundance of WsTDC transcripts reflected its preeminent role in withanamide biogenesis in the fruits. Transgenic lines overexpressing WsTDC gene showed accumulation of tryptamine at significantly higher levels, while lines silenced for WsTDC exhibited considerably depleted levels of tryptamine. Cloning and sequence analysis of promoter of WsTDC revealed the presence of W-box in it. Follow-up studies on isolation of WsWRKY1 transcription factor and its overexpression in W. somnifera revealed that WsTDC expression was substantially induced by WsWRKY1 resulting in overproduction of tryptamine. The study invokes a key role of TDC in regulating the indolyl secondary metabolites through enabling elevated flux/supply of tryptamine at multiple levels from gene expression to catalytic attributes overall coordinated by WsWRKY1. This is the first biochemical, molecular, structural, physiological and regulatory description of a fruit-functional TDC.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/genética , Proteínas de Plantas/genética , Triptaminas/biosíntesis , Withania/genética , Withania/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Clonación Molecular , Disacáridos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica de las Plantas , Indoles/metabolismo , Modelos Moleculares , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plantas Medicinales/genética , Plantas Medicinales/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triptaminas/metabolismo

RESUMEN

Radiation of the plant pyridoxal 5'-phosphate (PLP)-dependent aromatic l-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/química , Dominio Catalítico , Evolución Molecular , Proteínas de Plantas/química , Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidad por Sustrato

RESUMEN

Aromatic L-amino acid decarboxylase deficiency (AADCD) is a rare neurotransmitter metabolic disorder caused by DDC gene mutations, which leads to the metabolic disturbance of dopamine and serotonin. Most of the reported cases came from Taiwan China, but patients from mainland China were seldomly reported. The current study was the largest AADCD patient cohort from mainland China. Twenty-three patients with clinical features of AADCD and DDC gene variants were recruited. A total of 16 DDC variants were identified in this study, of which four variants (c.2T>C, c.277A>G, c.1021+1G>A, c.565G>T) were never reported previously. The intronic variant c.714+4A>T was the most common one, with an allele frequency of 45.7%. And patients carried this intronic variant presented with severe clinical manifestations, all of whom were bedridden. In this study, the average onset age was 3.61 ± 1.28 months and the average age of diagnosis was 12.91 ± 5.62 months. Early onset hypotonia, oculogyric crises, and autonomic symptoms such as excessive sweating, nasal congestion and profuse nasal, and oropharyngeal secretions, were common in our patients. Eighteen patients (78.3%) got various degree of improvement after using pyridoxine monotherapy or different combination of pyridoxine, dopamine agonists, and monoamine oxidase (MAO) inhibitors.

Asunto(s)

Errores Innatos del Metabolismo de los Aminoácidos/epidemiología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Descarboxilasas de Aminoácido-L-Aromático/deficiencia , Predisposición Genética a la Enfermedad/genética , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/fisiopatología , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , China , Estudios de Cohortes , Demografía , Agonistas de Dopamina/uso terapéutico , Exones , Femenino , Variación Genética , Heterocigoto , Homocigoto , Humanos , Lactante , Intrones , Masculino , Inhibidores de la Monoaminooxidasa/uso terapéutico , Hipotonía Muscular/complicaciones , Hipotonía Muscular/genética , Mutación , Piridoxina/uso terapéutico , Secuenciación del Exoma

RESUMEN

BACKGROUND: Aromatic L-amino acid decarboxylase deficiency (AADCD) is a rare, autosomal recessive inherited disorder which is characterized by neurological and vegetative symptoms. To date, only 130 patients with AADCD have been reported worldwide. METHODS: We demonstrated 14 previously undescribed patients together with three reportedly patients in Mainland China. Full clinical information was collected, and disease-causing variants in the DDC gene were detected. RESULTS: The common clinical manifestation of patients, including intermittent oculogyric crises, retarded movement development, and autonomic symptoms. Notably, a patient showed bone-density loss which have not been reported and two mildly phenotype patients improved psychomotor function after being prescribed medication. The most common genotype of Mainland Chinese AADCD is the splice-site variant (IVS6+4A> T; c.714+4A> T), which accounts for 58.8%, followed by c.1234C>T variant. Three novel compound heterozygous variants, c. 565G>T, c.170T>C, and c.1021+1G>A, were firstly reported. It is important to recognize the milder phenotypes of the disease as these patients might respond well to therapy. Besides, we discovered that patients may presented with milder if found to be compound heterozygote or homozygote for one of the following variants c.478C>G, c.853C>T, c.1123C>T, c.387G>A, and c.665T>C. DISCUSSION: The clinical data of the cohort of 17 patients in Mainland China broaden the clinical, molecular, and treatment spectrum of aromatic L-amino acid decarboxylase deficiency.

Asunto(s)

Errores Innatos del Metabolismo de los Aminoácidos/genética , Descarboxilasas de Aminoácido-L-Aromático/deficiencia , Descarboxilasas de Aminoácido-L-Aromático/genética , Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/patología , Descarboxilasas de Aminoácido-L-Aromático/química , Femenino , Heterocigoto , Humanos , Lactante , Masculino , Mutación , Fenotipo , Empalme del ARN

RESUMEN

AADC deficiency is a rare genetic disease caused by mutations in the gene of aromatic amino acid decarboxylase, the pyridoxal 5'-phosphate dependent enzyme responsible for the synthesis of dopamine and serotonin. Here, following a biochemical approach together with an in silico bioinformatic analysis, we present a structural and functional characterization of 13 new variants of AADC. The amino acid substitutions are spread over the entire protein from the N-terminal (V60A), to its loop1 (H70Y and F77L), to the large domain (G96R) and its various motifs, i.e. loop2 (A110E), or a core ß-barrel either on the surface (P210L, F251S and E283A) or in a more hydrophobic milieu (L222P, F237S and W267R) or loop3 (L353P), and to the C-terminal domain (R453C). Results show that the ß-barrel variants exhibit a low solubility and those belonging to the surface tend to aggregate in their apo form, leading to the identification of a new enzymatic phenotype for AADC deficiency. Moreover, five variants of residues belonging to the large interface of AADC (V60A, G96R, A110E, L353P and R453C) are characterized by a decreased catalytic efficiency. The remaining ones (H70Y and F77L) present features typical of apo-to-holo impaired transition. Thus, defects in catalysis or in the acquirement of the correct holo structure are due not only to specific local domain effects but also to long-range effects at either the protein surface or the subunit interface. Altogether, the new characterized enzymatic phenotypes represent a further step in the elucidation of the molecular basis for the disease.

Asunto(s)

Errores Innatos del Metabolismo de los Aminoácidos/genética , Descarboxilasas de Aminoácido-L-Aromático/deficiencia , Fenotipo , Algoritmos , Secuencias de Aminoácidos , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Catálisis , Biología Computacional , Escherichia coli , Variación Genética , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Mutación , Dominios Proteicos , Dispersión de Radiación , Solubilidad , Espectrofotometría , Relación Estructura-Actividad , Temperatura

RESUMEN

Schizophrenia is a major debilitating disorder worldwide. Schizophrenia is a result of multi-gene mutation and psycho-social factors. Mutated amino acid sequences in genes of DOPA such as TH, DDC, DBH, VMAT2, and NMDA (SET-1) have been implicated as major factors causing schizophrenia. In addition mutations in genes other than the DOPA genes such as RGS4, NRG1, COMT, AKT1 and DTNBP1 (SET 2) have also been implicated in the pathogenesis of schizophrenia. Several medicinal herbs and their bioactive constituents have been reported to be involved in ameliorating different neurological disorders including schizophrenia. The present study is mainly focused to study the effect of bioactive compound isolated from the celastrus panuculatus on DOPA and other related genes of schizophrenia using in silico approach. Moledular docking study was carriedout aginast all the selected targets with the lingds i.e. compound and clozapine using the autodock vina 4.0 module implemented in Pyrx 2010.12. The 3 D structures of genes of intrest were retrieved from the protein data bank (PDB). The bioavailability and pharmacological properties of the ligands were determined using OSIRIS server. The novelty of the compound was determined based on fitness, docking and bioavailability score. From the results it is observed that, the compoud has exhibited best dock score against all the selected targets than the clozapie except DBH and VMAT2 in SET-1 targets of DOPA genes. Where as the compound has shown best pharmacokinetic and biologicl property score than the clozapine. Hence, the compound can be considered for further in vitro and in vivo studies to determine the therapeutic efficacy and drug candidacy of the compound in future.

Asunto(s)

Dihidroxifenilalanina/antagonistas & inhibidores , Cetonas/farmacocinética , Extractos Vegetales/química , Propano/farmacología , Esquizofrenia/genética , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/efectos de los fármacos , Disponibilidad Biológica , Celastrus/química , Chalconas , Clozapina/química , Simulación por Computador , Bases de Datos de Proteínas , Dihidroxifenilalanina/genética , Humanos , Cetonas/uso terapéutico , Ligandos , Conformación Molecular , Simulación del Acoplamiento Molecular/métodos , Mutación/genética , N-Metilaspartato/antagonistas & inhibidores , N-Metilaspartato/química , Extractos Vegetales/farmacología , Propano/análogos & derivados , Esquizofrenia/tratamiento farmacológico

RESUMEN

Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Escherichia coli/metabolismo , Proteínas de Insectos/metabolismo , Ingeniería Metabólica/métodos , Tetrahidropapaverolina/metabolismo , Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Ácido 3,4-Dihidroxifenilacético/metabolismo , Secuencias de Aminoácidos/genética , Animales , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/aislamiento & purificación , Bombyx , Dopamina/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad

RESUMEN

In a previous work, we in silico annotated protein sequences of Citrus genus plants as putative tryptophan decarboxylase (pTDC). Here, we investigated the structural properties of Citrus pTDCs by using the TDC sequence of Catharanthus roseus as an experimentally annotated reference to carry out comparative modeling and substrate docking analyses. The functional annotation as TDC was verified by combining 3D molecular modeling and docking simulations, evidencing the peculiarities and the structural similarities with C. roseus TDC. Docking with l-tryptophan as a ligand showed specificity of pTDC for this substrate. These combined results confirm our previous in silico annotation of the examined protein sequences of Citrus as TDC and provide support for TDC activity in this plant genus.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/química , Citrus/enzimología , Modelos Moleculares , Simulación del Acoplamiento Molecular , Triptófano/química , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Ligandos , Especificidad por Sustrato , Triptófano/metabolismo

RESUMEN

Kiwifruit (Actinidia deliciosa cv. Hayward) is a commercially important crop with highly nutritional green fleshy fruits. The post-harvest maturation of the fruits is well characterized, but little is known about the metabolic changes that occur during fruit development. Here we used untargeted metabolomics to characterize the non-volatile metabolite profile of kiwifruits collected at different time points after anthesis, revealing profound metabolic changes before the onset of ripening including the depletion of many classes of phenolic compounds. In contrast, the phytohormone abscisic acid accumulated during development and ripening, along with two indolamines (serotonin and its precursor tryptamine), and these were monitored in greater detail by targeted metabolomics. The role of indolamines in kiwifruit development is completely unknown, so we also characterized the identity of genes encoding tryptophan decarboxylase in A. deliciosa and its close relative A. chinensis to provide insight into the corresponding biological processes. Our results indicate that abscisic acid and indolamines fulfill unrecognized functions in the development and ripening of kiwifruits.

Asunto(s)

Actinidia/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Metaboloma , Metabolómica , Desarrollo de la Planta , Actinidia/clasificación , Actinidia/genética , Secuencia de Aminoácidos , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Cromatografía Liquida , Biología Computacional/métodos , Frutas/metabolismo , Espectrometría de Masas , Metabolómica/métodos , Filogenia , Desarrollo de la Planta/genética , Serotonina/metabolismo , Triptaminas/metabolismo

RESUMEN

L-Dopa decarboxylase (DDC) catalyzes the decarboxylation of L-Dopa to dopamine and 5-hydroxytryptophan (5-HTP) to serotonin. Although DDC has been purified from a variety of peripheral organs, including the liver, kidney and pancreas, the physiological significance of the peripherally expressed enzyme is not yet fully understood. DDC has been considered as a potential novel biomarker for various types of cancer, however, the role of DDC in the development of hepatocellular carcinoma (HCC) remains to be evaluated. Phosphatidylinositol 3-kinase (PI3K), on the other hand, has been shown to play a key role in the tumorigenesis, proliferation, metastasis, apoptosis, and angiogenesis of HCC by regulating gene expression. We initially identified the interaction of DDC with PI3K by means of the phage display methodology. This association was further confirmed in human hepatocellular carcinoma cell lines, human embryonic kidney cells, human neuroblastoma cells, as well as mouse brain, by the use of specific antibodies raised against DDC and PI3K. Functional aspects of the above interaction were studied upon treatment with the DDC inhibitor carbidopa and the PI3K inhibitor LY294002. Interestingly, our data demonstrate the expression of the neuronal type DDC mRNA in HCC cells. The present investigation provides new evidence on the possible link of DDC with the PI3K pathway, underlining the biological significance of this complex enzyme.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Carbidopa/farmacología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neuroblastoma/metabolismo , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Descarboxilasas de Aminoácidos Aromáticos/farmacología , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Biblioteca de Péptidos , Fosfatidilinositol 3-Quinasas/genética , Células Tumorales Cultivadas

RESUMEN

Aromatic l-amino acid decarboxylases (AAADs) are a phylogenetically diverse group of enzymes responsible for the decarboxylation of aromatic amino acid substrates into their corresponding aromatic arylalkylamines. AAADs have been extensively studied in mammals and plants as they catalyze the first step in the production of neurotransmitters and bioactive phytochemicals, respectively. Unlike mammals and plants, the hallucinogenic psilocybin mushroom Psilocybe cubensis reportedly employs an unrelated phosphatidylserine-decarboxylase-like enzyme to catalyze l-tryptophan decarboxylation, the first step in psilocybin biosynthesis. To explore the origin of this chemistry in psilocybin mushroom, we generated the first de novo transcriptomes of P. cubensis and investigated several putative l-tryptophan-decarboxylase-like enzymes. We report the biochemical characterization of a noncanonical AAAD from P. cubensis ( PcncAAAD) that exhibits substrate permissiveness toward l-phenylalanine, l-tyrosine, and l-tryptophan, as well as chloro-tryptophan derivatives. The crystal structure of PcncAAAD revealed the presence of a unique C-terminal appendage domain featuring a novel double-ß-barrel fold. This domain is required for PcncAAAD activity and regulates catalytic rate and thermal stability through calcium binding. PcncAAAD likely plays a role in psilocybin production in P. cubensis and offers a new tool for metabolic engineering of aromatic-amino-acid-derived natural products.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Calcio/metabolismo , Psilocybe/enzimología , Psilocibina/biosíntesis , Aminoácidos Aromáticos/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/genética , Dominio Catalítico , Catharanthus/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Papaver/enzimología , Filogenia , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Psilocybe/genética , Especificidad por Sustrato , Transcriptoma , Levaduras/genética

RESUMEN

Melatonin plays an important role in plant growth, development, and environmental stress. In this study, a systematic analysis of tomato tryptophan decarboxylase (SlTrpDC), which is the first enzyme of melatonin biosynthesis, was conducted by integrating structural features, phylogenetic relationships, an exon/intron feature, and a divergent expression profile. The results determined that the tomato genome encoded five members (SlTrpDC1-SlTrpDC5). The phylogenetic relationships indicated that gene expansion was proposed as the major mode of evolution of the TrpDC genes from the different plant algae species to the higher plants species. The analyses of the exon/intron configurations revealed that the intron loss events occurred during the structural evolution of the TrpDCs in plants. Additionally, the RNA-seq and qRT-PCR analysis revealed that the expression of the SlTrpDC3 was high in all of the tested tissues, while the SlTrpDC4 and SlTrpDC5 were not expressed. The expression patterns of the remaining two (SlTrpDC1 and SlTrpDC2) were tissue-specific, which indicated that these genes may play important roles within the different tissues. No expression difference was observed in the tomato plants in response to the biotic stresses. This study will expand the current knowledge of the roles of the TrpDC genes in tomato growth and development.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Secuencia de Aminoácidos , Descarboxilasas de Aminoácido-L-Aromático/química , Biología Computacional/métodos , Activación Enzimática , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Solanum lycopersicum/clasificación , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformación Proteica , Análisis de Secuencia de ADN , Relación Estructura-Actividad

RESUMEN

Aromatic amino acid or Dopa decarboxylase (AADC or DDC) is a homodimeric pyridoxal 5'-phosphate (PLP) enzyme responsible for the generation of the neurotransmitters dopamine and serotonin. AADC deficiency is a rare inborn disease caused by mutations of the AADC gene leading to a defect of AADC enzyme and resulting in impaired dopamine and serotonin synthesis. Until now, only the molecular effects of homozygous mutations were analyzed. However, although heterozygous carriers of AADC deficiency were identified, the molecular aspects of their enzymatic phenotypes are not yet investigated. Here, we focus our attention on the R347Q/R358H and R347Q/R160W heterozygous mutations, and report for the first time the isolation and characterization, in the purified recombinant form, of the R347Q/R358H heterodimer and of the R358H homodimer. The results, integrated with those already known of the R347Q homodimeric variant, provide evidence that (i) the R358H mutation strongly reduces the PLP-binding affinity and the catalytic activity, and (ii) a positive interallelic complementation exists between the R347Q and the R358H mutations. Bioinformatics analyses provide the structural basis for these data. Unfortunately, the R347Q/R160W heterodimer was not obtained in a sufficient amount to allow its purification and characterization. Nevertheless, the biochemical features of the R160W homodimer give a contribution to the enzymatic phenotype of the heterozygous R347Q/R160W and suggest the possible relevance of Arg160 in the proper folding of human DDC. © 2018 IUBMB Life, 70(3):215-223, 2018.

Asunto(s)

Errores Innatos del Metabolismo de los Aminoácidos/embriología , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/deficiencia , Multimerización de Proteína/genética , Proteínas Recombinantes/química , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Catálisis , Dopamina/biosíntesis , Heterocigoto , Humanos , Mutación , Pliegue de Proteína , Proteínas Recombinantes/genética , Serotonina/biosíntesis

RESUMEN

In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO2, NH3, and H2O2. This contrasts to the typical DDC-catalyzed reaction, which produces CO2 and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H2O2 in the process. Biochemical assessment established that H2O2, formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H2O; thereby avoiding oxidative stress by H2O2. Results of our structural and functional analyses provide a reasonable explanation of mechanisms involved in DHPA synthase-mediated reactions. Based on the key active site residue Asn192, identified in Drosophila DHPA synthase, we were able to distinguish all available insect DHPA synthases from DDC sequences primarily.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/aislamiento & purificación , Proteínas de Drosophila/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Dominio Catalítico , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo

RESUMEN

Human histidine decarboxylase (HDC) and dopa decarboxilase (DDC) are highly homologous enzymes responsible for the synthesis of biogenic amines (BA) like histamine, and serotonin and dopamine, respectively. The enzymes share many structural and functional analogies, while their product metabolisms also follow similar patterns that are confluent in some metabolic steps. They are involved in common physiological functions, such as neurotransmission, gastrointestinal track function, immunity, cell growth and cell differentiation. As a consequence, metabolic elements of both BA subfamilies are also co-participants in a long list of human diseases. This review summarizes the analogies and differences in their origin (HDC and DDC) as well as their common pathophysiological scenarios. The major gaps of information are also underlined, as they delay the possibility of holistic approaches that would help personalized medicine and pharmacological initiatives for prevalent and rare diseases.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Histidina Descarboxilasa/metabolismo , Animales , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Dopamina/metabolismo , Histamina/metabolismo , Histidina Descarboxilasa/química , Histidina Descarboxilasa/genética , Humanos , Modelos Moleculares , Enfermedades Raras/genética , Enfermedades Raras/metabolismo , Serotonina/metabolismo

RESUMEN

Aromatic L-amino acid decarboxylases (AADCs) catalyze the release of CO2 from proteinogenic and non-proteinogenic L-amino acid substrates and are involved in pathways that biosynthesize neurotransmitters or bioactive natural products. In contrast to AADCs from animals and plants, fungal AADCs have received very little attention. Here, we report on the in vitro characterization of heterologously produced Ceriporiopsis subvermispora AADC, now referred to as CsTDC, which is the first characterized basidiomycete AADC. This study identified the enzyme as a decarboxylase that is strictly specific for L-tryptophan and 5-hydroxy-L-tryptophan. The tdc gene was subjected to saturation mutagenesis so as to vary the key active site residue, Gly351. Aliphatic amino acid residues, L-serine, or L-threonine at position 351 added L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA) decarboxylase activity while retaining stereospecificity and L-tryptophan decarboxylase activity.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/química , Basidiomycota/enzimología , Ingeniería de Proteínas , Descarboxilasas de Aminoácido-L-Aromático/genética , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Variación Genética , Estructura Molecular , Especificidad por Sustrato