Aromatic L-amino acid decarboxylases: mechanistic features and microbial applications.

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.

Aromatic Amino Acid Decarboxylase Deficiency: The Added Value of Biochemistry.

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.

Cloning, characterization, and enzymatic identification of a new tryptophan decarboxylase from Ophiorrhiza pumila.

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.

WRKY1-mediated regulation of tryptophan decarboxylase in tryptamine generation for withanamide production in Withania somnifera (Ashwagandha).

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.

Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins.

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.

The genetic and clinical characteristics of aromatic L-amino acid decarboxylase deficiency in mainland China.

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.

Aromatic L-amino acid decarboxylase deficiency in 17 Mainland China patients: Clinical phenotype, molecular spectrum, and therapy overview.

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.

New variants of AADC deficiency expand the knowledge of enzymatic phenotypes.

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.

Bioavailability and Neuroprotectivity of 3-(3, 4-dimethoxy phenyl)-1-4 (methoxy phenyl) prop-2-en-1-one against Schizophrenia: an in silico approach.

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.

Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

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.

Structure and Ligands Interactions of Citrus Tryptophan Decarboxylase by Molecular Modeling and Docking Simulations.

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.

Untargeted and Targeted Metabolomics and Tryptophan Decarboxylase In Vivo Characterization Provide Novel Insight on the Development of Kiwifruits (Actinidia deliciosa).

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.

L-Dopa decarboxylase interaction with the major signaling regulator ΡΙ3Κ in tissues and cells of neural and peripheral origin.

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.

Monoamine Biosynthesis via a Noncanonical Calcium-Activatable Aromatic Amino Acid Decarboxylase in Psilocybin Mushroom.

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.

The Tryptophan Decarboxylase in Solanum lycopersicum.

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.

Heterozygosis in aromatic amino acid decarboxylase deficiency: Evidence for a positive interallelic complementation between R347Q and R358H mutations.

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.

Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions.

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.

Structural and functional analogies and differences between histidine decarboxylase and aromatic l-amino acid decarboxylase molecular networks: Biomedical implications.

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.

Active-Site Engineering Expands the Substrate Profile of the Basidiomycete L-Tryptophan Decarboxylase CsTDC.

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.

Carbidopa-based modulation of the functional effect of the AAV2-hAADC gene therapy in 6-OHDA lesioned rats.

Progressively blunted response to L-DOPA in Parkinson's disease (PD) is a critical factor that complicates long-term pharmacotherapy in view of the central importance of this drug in management of the PD-related motor disturbance. This phenomenon is likely due to progressive loss of one of the key enzymes involved in the biosynthetic pathway for dopamine in the basal ganglia: aromatic L-amino acid decarboxylase (AADC). We have developed a gene therapy based on an adeno-associated virus encoding human AADC (AAV2-hAADC) infused into the Parkinsonian striatum. Although no adverse clinical effects of the AAV2-hAADC gene therapy have been observed so far, the ability to more precisely regulate transgene expression or transgene product activity could be an important long-term safety feature. The present study was designed to define pharmacological regulation of the functional activity of AAV2-hAADC transgene product by manipulating L-DOPA and carbidopa (AADC inhibitor) administration in hemi-parkinsonian rats. Thirty days after unilateral striatal infusion of AAV2-hAADC, animals displayed circling behavior and acceleration of dopamine metabolism in the lesioned striatum after administration of a low dose of L-DOPA (5 mg/kg) co-administered with 1.25 mg/kg of carbidopa. This phenomenon was not observed in control AAV2-GFP-treated rats. Withdrawal of carbidopa from a daily L-DOPA regimen decreased the peripheral L-DOPA pool, resulting in almost total loss of L-DOPA-induced behavioral response in AAV2-hAADC rats and a significant decline in striatal dopamine turnover. The serum L-DOPA level correlated with the magnitude of circling behavior in AAV2-hAADC rats. Additionally, AADC activity in homogenates of lesioned striata transduced by AAV2-AADC was 10-fold higher when compared with AAV2-GFP-treated control striata, confirming functional transduction. Our data suggests that the pharmacological regulation of circulating L-DOPA might be effective in the controlling of function of AAV2-hAADC transgene product in PD gene therapy.