Aromatic L-amino Acid Decarboxylase Research Articles

In silico Evaluation of the Feasibility of Magnolia officinalis Electron-shuttling Compounds as Parkinson’s Disease Remedy

Background: Parkinson’s Disease is one of the leading neurodegenerative disorders in the world. Currently, there is still no treatment that could completely cure the disease. Traditional Chinese Medicine has been a source for drug candidates, and many studies have elucidated its pharmacokinetic capabilities. Previous studies showed that Magnolia officinalis has anti-inflammatory, antioxidant, and bioenergy generation activities. Furthermore, the electron-shuttling and bioenergy-stimulating capabilities of herbal and brain disorder medicines have been linked to their effectiveness as a remedy. Objective: This preliminary study aims to evaluate the electron-shuttling compounds of Magnolia officinalis (i.e., acteoside, isoquercitrin, magnatriol B, obovatol, quercitrin, randaiol, and rutin) as potential drug candidates for Parkinson’s Disease. Methods: The seven electron-shuttling compounds were individually docked to the five Parkinson’s Disease-related proteins, namely aromatic L-amino acid decarboxylase, α-synuclein, monoamine oxidase B, catechol-o-methyltransferase, and A2A adenosine receptor, using LibDock. ADMET predictions were also made to screen the compounds further. Results: Molecular docking results showed that all compounds have relatively high LibDock scores against the proteins, with acteoside, isoquercitrin, and rutin having the highest scores. However, considering the ADMET results, only magnatriol B, obovatol, and randaiol had optimal properties as candidates for neurodegenerative drugs. Conclusion: The electron-shuttling compounds of M. officinalis, magnatriol B, obovatol, and randaiol, have the potential to be a remedy for Parkinson’s Disease due to their high probability of binding to the proteins.

Sustainable production of the drug precursor tyramine by engineered Corynebacterium glutamicum.

Tyramine has attracted considerable interest due to recent findings that it is an excellent starting material for the production of high-performance thermoplastics and hydrogels. Furthermore, tyramine is a precursor of a diversity of pharmaceutically relevant compounds, contributing to its growing importance. Given the limitations of chemical synthesis, including lack of selectivity and laborious processes with harsh conditions, the biosynthesis of tyramine by decarboxylation of L-tyrosine represents a promising sustainable alternative. In this study, the de novo production of tyramine from simple nitrogen and sustainable carbon sources was successfully established by metabolic engineering of theL-tyrosine overproducing Corynebacterium glutamicum strain AROM3. A phylogenetic analysis of aromatic-L-amino acid decarboxylases (AADCs) revealed potential candidate enzymes for the decarboxylation of tyramine. The heterologous overexpression of the respective AADC genes resulted in successful tyramine production, with the highest tyramine titer of 1.9gL-1 obtained for AROM3 overexpressing the tyrosine decarboxylase gene of Levilactobacillus brevis. Further metabolic engineering of this tyramine-producing strain enabled tyramine production from the alternative carbon sources ribose and xylose. Additionally, up-scaling of tyramine production from xylose to a 1.5L bioreactor batch fermentation was demonstrated to be stable, highlighting the potential for sustainable tyramine production. KEY POINTS: • Phylogenetic analysis revealed candidate l-tyrosine decarboxylases • C. glutamicum was engineered for de novo production of tyramine • Tyramine production from alternative carbon substrates was enabled.

Transcriptomic Analysis Reveals the Molecular Defense Mechanisms of Poa pratensis Against Powdery Mildew Fungus Blumeria graminis f. sp. Poae

Kentucky bluegrass (Poa pratensis L.) is a valuable cool-season turfgrass widely utilized for forage, turf, and ecological purposes; however, its productivity and ornamental value are significantly compromised by powdery mildew, caused by Blumeria graminis f. sp. Poae, which negatively affects turf quality. In the present study, we examined the interactions between P. pratensis varieties and B. graminis, focusing on primary haustorium formation at 24 h post-inoculation and the formation of germ tubes at 48 h post-inoculation. We explored the molecular mechanisms underlying the response of different P. pratensis varieties at 48 h post-inoculation via transcriptomic techniques. Our results revealed that the primary haustorium formation rate in ‘Taihang’ at 24 h after B. graminis inoculation was significantly lower than that in ‘Explorer’ and ‘Black Jack’. The conidia of B. graminis could form two to five germ tubes, and the proportion of conidia that formed five germ tubes in ‘Taihang‘ at 48 h post-inoculation was significantly lower than that in the other two varieties. Transcriptome analysis revealed 680,765 transcripts as unigenes. A total of 9983 unigenes were identified as differentially expressed genes in one or more varieties of P. pratensis after inoculation with powdery mildew compared with the control. In total, 6284 differentially expressed genes were upregulated in ‘Taihang’, which was substantially greater than those in ‘Black Jack’ (4530) and ‘Explorer’ (4666). Moreover, 2843 differentially expressed genes were specific to ‘Taihang’, whereas 1644 and 1590 unique differentially expressed genes were specific to ‘Explorer’ and ‘Black Jack’, respectively. The specifically expressed genes play crucial roles in determining the disease resistance of powdery mildew. Notably, the expression of genes encoding chitinase, gamma-glutamyltranspeptidase 1, UDP-arabinopyranose mutase 1, oxalate oxidase 2, mitogen-activated protein kinase kinase 1-like, tryptophan decarboxylase, and aromatic L-amino acid decarboxylase was closely related to powdery mildew resistance in ‘Taihang’, making them promising candidate genes for studying resistance to powdery mildew in P. pratensis. This study identified critical genes involved in powdery mildew resistance in P. pratensis, providing a basis for future gene mining and molecular breeding to increase disease resistance in P. pratensis.

Optimizing the anesthetic care of patients with aromatic l-amino acid decarboxylase deficiency.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disorder that results in a lack of the monoamine neurotransmitters dopamine, serotonin, norepinephrine, and epinephrine. Patients present with a wide spectrum of symptoms, including motor and autonomic dysfunction, hypotonia, and developmental delay, often before the age of one. Until recently, treatment options were limited to symptom control, but the recent approval of the first gene therapy for AADC deficiency in Europe and the UK has provided an alternative to treating symptoms for this disease. Eladocagene exuparvovec is a one-time gene therapy, administered bilaterally to the putamen by magnetic resonance imaging-guided stereotactic neurosurgery. While administration of the gene therapy itself is minimally invasive, the anesthetic management of patients with AADC deficiency is challenging due to the absence of sympathetic regulation secondary to the lack of adrenergic neurotransmitters. Optimal anesthetic management requires an understanding of the complex and heterogeneous nature of the disease. Hemodynamic instability, temperature dysregulation, and hypoglycemia are of primary concern, but there are also challenges regarding intravenous access and airway management. A thorough preoperative assessment is essential and should be guided by the patient's history. Advanced planning is necessary regarding the timing of the procedure schedule and operative plan; meticulous preparation, simulation for the operating room, as well as communication with all perioperative staff members, are crucial. Intraoperatively, utmost care must be taken to protect the skin, maintain body temperature, and to prepare for inotropic and/or glycemic support as needed. Postoperative intensive care management is necessary for consideration of postoperative extubation and provision of supportive care. With careful planning, preparation, and vigilance, patients with AADC deficiency can safely undergo anesthesia.

Nigrostriatal tract defects in mice with aromatic l-amino acid decarboxylase deficiency

The development of the nigrostriatal dopaminergic (DA) pathway in the brain involves many transcriptional and chemotactic molecules, and a deficiency of these molecules can cause nigrostriatal tract defects. However, the role of the end product, dopamine, in nigrostriatal pathway development has not been described. In the present study, we analyzed a mouse model of congenital dopamine and serotonin deficiency, namely, the aromatic l-amino acid decarboxylase (AADC) deficiency (DdcKI) mouse model. We found via tyrosine hydroxylase (TH) immunofluorescence staining that the number of DA fibers in the stratum of 14-day-old DdcKI mice decreased. In TH-stained cleared whole brains of DdcKI mice, the numbers of DA neurons in the substantia nigra (SN) and the number of DA nerve bundles leaving the SN were both normal. However, we found that the nigrostriatal bundles in DdcKI mice were dispersed, taking aberrant routes to the striatum and spreading over a wide area. The total volume occupied by the nigrostriatal tract was increased, and the fraction of TH staining in the tract was decreased in DdcKI mice. Single-nucleus RNA sequencing analysis for mice 0, 7, and 14 days of age, revealed delayed axonogenesis and synapse formation in the striatum of DdcKI mice. The CellChat program inferred less cell–cell communication between striatal D1/D2 neurons but increased cell–cell communication involving neural precursors in DdcKI mice. Therefore, a congenital deficiency in dopamine affects nigrostriatal axon extension and striatal innervation. These nigrostriatal tract defects may limit the treatment efficacy for patients with TH or AADC deficiency.

MC-LR disrupts dopamine synthesis in the substantia nigra of midbrain by enhancing the chaperone-mediated autophagy pathway through direct binding to ERK2

Microcystins are environmental toxins produced by freshwater cyanobacteria. Microcystin-LR (MC-LR) is one of the most abundant and harmful isomers. MC-LR poses a serious threat to human health. MC-LR could penetrate the blood-brain barrier of mice and accumulate in the substantia nigra (SN) of the midbrain, leading to a reduction in dopamine levels and Parkinson's disease (PD)-like motor dysfunction in mice. The reduction in dopamine levels is a key factor contributing to movement disorders in humans with PD. Dopamine is synthesized in the dopaminergic neurons of the SN by the actions of tyrosine hydroxylase (TH) and dihydroxyphenylalanine decarboxylase (DDC). In this study, we found that MC-LR could enter dopaminergic neurons in the SN and directly bound to extracellular signal-regulated kinase 2 (ERK2), enhancing ERK2 stability. ERK2 further enhanced the transcriptional activity of Heat Shock Protein Family A Member 8 (HSPA8) and promoted the expression of Heat shock cognate 71 kDa protein (HSC70), which in turn amplified the chaperone-mediated autophagy (CMA) pathway and accelerated the degradation of TH and DDC. This affected the dopamine synthesis process, resulting in a significant reduction in dopamine levels. The study is the first to reveal that ERK2 was a direct target of MC-LR, and further enhanced CMA affecting dopamine synthesis, which has important theoretical and practical significance for environmental safety management.

Investigating the Correlation between Dopa Decarboxylase (DDC) and Dopamine Beta-Hydroxylase (DBH) genes Expression in Parkinson's and schizophrenia patients

Investigating the Correlation between Dopa Decarboxylase (DDC) and Dopamine Beta-Hydroxylase (DBH) genes Expression in Parkinson's and schizophrenia patients

Biofluid Markers and Tissue Biopsies Analyses for the Prodromal and Earliest Phase of Parkinson's Disease.

The recent development of new methods to detect misfolded α-synuclein (αSyn) aggregates in biofluids and tissue biopsies in the earliest Parkinson's disease (PD) phases is dramatically challenging the biological definition of PD. The αSyn seed amplification methods in cerebrospinal fluid (CSF) showed high sensitivity and specificity for early diagnosis of PD and Lewy bodies disorders. Several studies in isolated REM sleep behavior disorders and other at-risk populations also demonstrated a high prevalence of CSF αSyn positivity and its potential value in predicting the phenoconversion to clinically manifested diseases. Growing evidence exists for αSyn aggregates in olfactory mucosa, skin, and other tissues in subjects with PD or at-risk subjects. DOPA decarboxylase and numerous other candidates have been additionally proposed for either diagnostic or prognostic purposes in earliest PD phases. The newly described αSyn detection in blood, through its quantification in neuronally-derived exosome vesicles, represents a technical challenge that could open a new scenario for the biological diagnosis of PD. Despite this growing evidence in the field, most of method of αSyn detection and markers still need to be validated in ongoing longitudinal studies through an accurate assessment of different prodromal disease subtypes and scenarios before being definitively implemented in clinical settings.

Immunohistochemical analysis and distribution of epithelial mast cells in the rat larynx and trachea.

Mast cells (MCs) in rat airways have been classified into two subtypes: epithelial MCs and connective tissue MCs (CTMCs). However, the immunohistochemical characteristics, cellular morphology, and distribution of epithelial MCs in the upper airways remain unclear. The present study investigated the morphological characteristics and distribution of epithelial MCs using 5-hydroxytryptamine (5-HT) and other immunohistochemical markers in sectioned or whole-mount preparations of the rat larynx and trachea. A double immunofluorescence analysis revealed the colocalization of 5-HT immunoreactivity with c-kit, a stem cell factor receptor commonly used as a MC marker, in both epithelial MCs and CTMCs. Dopa decarboxylase, an enzyme involved in 5-HT synthesis, was detected in both subtypes, suggesting their ability to synthesize and release 5-HT. Tryptase and histidine decarboxylase (a biosynthetic enzyme of histamine), which are well-known mediators of MCs, were exclusive to CTMCs. Epithelial MCs were pleomorphic with long cytoplasmic processes, whereas CTMCs were round and lacked cytoplasmic processes. The density of epithelial MCs was significantly higher in the glottis and cranial part of the trachea than in the epiglottis and other parts of the trachea. The present results showed that the morphology and immunohistochemical characteristics of epithelial MCs were different from those of CTMCs in the rat larynx and trachea, and variform epithelial MCs were predominantly located at the entrance of the upper airways. Epithelial MCs may release 5-HT to regulate innate immune responses by modulating epithelial cell functions at the entrance gate of the upper airways.

The Catalytic Mechanism of Decarboxylation of L‐DOPA to Dopamine by Papaver Somniferum Aromatic Amino Acid Decarboxylase ‐ A Computational Study

The Catalytic Mechanism of Decarboxylation of L‐DOPA to Dopamine by Papaver Somniferum Aromatic Amino Acid Decarboxylase ‐ A Computational Study

Structure and evolution of alanine/serine decarboxylases and the engineering of theanine production.

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine.

Structure and evolution of alanine/serine decarboxylases and the engineering of theanine production

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine.

Mild/moderate phenotypes in AADC deficiency: Focus on the aromatic amino acid decarboxylase protein.

AADC deficiency is a severe neurometabolic inherited rare disorder due to the absence or decrease of dopamine and serotonin levels, causing deep motor and neurodevelopmental impairments. The disease is often fatal in the first decade of life, and pharmacological treatments (dopamine agonists, pyridoxine, and monoamine oxidase inhibitors as the first-line choices) can only alleviate the symptoms. Gene therapy surgery is now available for severe patients in the European Union and the United Kingdom, and follow-up data witness encouraging improvements. In the past few years, mostly due to the increased awareness and knowledge of AADC deficiency, together with newborn screening programs and advancements in methods for genetic diagnosis, the number of mild/moderate phenotypes of AADC deficiency patients has increased to 12% of the total. A review of the genotypes (homozygous/compound heterozygous) of AADC deficiency mild/moderate patients is presented here. The pathogenicity classification of each genetic variant is discussed. Then, we focused on the type of AADC protein possessed by patients and on the predictable structural score of the homodimeric/heterodimeric species of each protein variant. Since the terminology used for genetic and protein variants is the same, we highlighted how it could be misleading. We analyzed the loss-of-function as a fold-change decrease of activity of the recombinant purified AADC enzyme(s) theoretically synthesized by mild/moderate patients. A minimal residual kcat of 8% and/or kcat/Km of 1% seems necessary to avoid a severe disease manifestation. Overall, this cluster of mild/moderate patients needs consideration for a more appropriate and aimed therapeutic approach.

Benserazide is neuroprotective and improves functional recovery after experimental ischemic stroke by altering the immune response

Stroke is a leading cause of permanent disability worldwide. Despite intensive research over the last decades, key anti-inflammatory strategies that have proven beneficial in pre-clinical animal models have often failed in translation. The importance of neutrophils as pro- and anti-inflammatory peripheral immune cells has often been overlooked in ischemic stroke. However, neutrophils rapidly infiltrate into the brain parenchyma after stroke and secrete an array of pro-inflammatory factors including reactive oxygen species, proteases, cytokines, and chemokines exacerbating damage. In this study, we demonstrate the neuroprotective and anti-inflammatory effect of benserazide, a clinically used DOPA decarboxylase inhibitor, using both in vitro models of inflammation and in vivo mouse models of focal cerebral ischemia. Benserazide significantly attenuated PMA-induced NETosis in isolated human neutrophils. Furthermore, benserazide was able to protect both SH-SY5Y and iPSC-derived human cortical neurons when challenged with activated neutrophils demonstrating the clinical relevance of this study. Additional in vitro data suggest the ability of benserazide to polarize macrophages towards M2-phenotypes following LPS stimulation. Neuroprotective effects of benserazide are further demonstrated by in vivo studies where peripheral administration of benserazide significantly attenuated neutrophil infiltration into the brain, altered microglia/macrophage phenotypes, and improved the behavioral outcome post-stroke. Overall, our data suggest that benserazide could serve as a drug candidate for the treatment of ischemic stroke. The importance of our results for future clinical trials is further underlined as benserazide has been approved by the European Medicines Agency as a safe and effective treatment in Parkinson’s disease when combined with levodopa.

Potential Therapeutic Approach using Aromatic l-amino Acid Decarboxylase and Glial-derived Neurotrophic Factor Therapy Targeting Putamen in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative illness characterized by specific loss of dopaminergic neurons, resulting in impaired motor movement. Its prevalence is twice as compared to the previous 25 years and affects more than 10 million individuals. Lack of treatment still uses levodopa and other options as disease management measures. Treatment shifts to gene therapy (GT), which utilizes direct delivery of specific genes at the targeted area. Therefore, the use of aromatic L-amino acid decarboxylase (AADC) and glial-derived neurotrophic factor (GDNF) therapy achieves an effective control to treat PD. Patients diagnosed with PD may experience improved therapeutic outcomes by reducing the frequency of drug administration while utilizing provasin and AADC as dopaminergic protective therapy. Enhancing the enzymatic activity of tyrosine hydroxylase (TH), glucocorticoid hormone (GCH), and AADC in the striatum would be useful for external L-DOPA to restore the dopamine (DA) level. Increased expression of glutamic acid decarboxylase (GAD) in the subthalamic nucleus (STN) may also be beneficial in PD. Targeting GDNF therapy specifically to the putaminal region is clinically sound and beneficial in protecting the dopaminergic neurons. Furthermore, preclinical and clinical studies supported the role of GDNF in exhibiting its neuroprotective effect in neurological disorders. Another Ret receptor, which belongs to the tyrosine kinase family, is expressed in dopaminergic neurons and sounds to play a vital role in inhibiting the advancement of PD. GDNF binding on those receptors results in the formation of a receptor-ligand complex. On the other hand, venous delivery of recombinant GDNF by liposome-based and encapsulated cellular approaches enables the secure and effective distribution of neurotrophic factors into the putamen and parenchyma. The current review emphasized the rate of GT target GDNF and AADC therapy, along with the corresponding empirical evidence.

Extraction of melanin from the shell and mantle and activities of enzymes related to the melanin biosynthesis in the Ruditapes philippinarum

The shell surface of Manila clam (Ruditapes philippinarum) has a variety of colors and stripes, with diverse shell color polymorphism. As an important trait of economic shellfish, shell color plays an important role in the selection breeding of shellfish species. In this study, we extracted melanin from the shell and mantle of R. philippinarum by the hydrochloric acid method and measured the melanin content by spectrophotometry. The absorption spectrum of melanin and its absorption peak at 350 nm absorbance was determined. We found significant differences in the melanin content of clams with dark strain and light strain, as well as four different shell colors. Moreover, we further detected the activities of enzymes related to melanin synthesis in R. philippinarum mantle, such as tyrosinase (TYR), tyrosine hydroxylase (TH), dopa decarboxylase (DDC), N-acetyltransferase (NAT), and N-β- alanyldopamine (NBAD) in the mantle of R. philippinarum. The results showed that the activities of TYR, TH, DDC and NAT were positively correlated with the content of melanin. However, the activity of NBAD was negatively correlated with the content of melanin. Altogether, these results indicating that these enzymes are involved in the regulation of the melanogenesis pathway of R. philippinarum. This study provides valuable information for further research on shell color genetic mechanism and breeding of R. philippinarum.

Emerging Gene Therapies for Alzheimer's and Parkinson's Diseases: An Overview of Clinical Trials and Promising Candidates.

Gene therapy as a disease-modifying therapeutic approach for neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), is a promising avenue. Promising results in the preclinical studies involving rodents and nonhuman primates utilizing gene therapy have led to multiple clinical trials evaluating various genes of interest for AD and PD. In AD, clinical trials are assessing gene therapy involving brain-derived neurotrophic factor (BDNF) and other targets such as apolipoprotein E2 (APOE2) and human telomerase reverse transcriptase (hTERT). In PD, clinical trials are evaluating gene therapy delivering neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF). Additionally, gene therapy delivering enzymes aromatic L-amino acid decarboxylase (AADC) and glutamic acid decarboxylase (GAD) are also being evaluated for PD. All these trials primarily utilized adeno-associated virus (AAV) to deliver the above transgene of interest. This review summarizes the current clinical trials involving gene therapy for AD and PD. It also discussesthe challenges and opportunities associated with the gene therapy approach in AD and PD and ongoing developments related to increasing the safety and efficacy of the gene therapy for long-term outcomes, which include evaluation of various serotypes and administration routes. This comprehensive review emphasizes translating preclinical findings into clinical trials, further directions, and the potential for this promising therapeutic approach to alleviate neurodegenerative disease.

Morphometric analysis and functional insights into the serotonergic system of Girardia tigrina (Tricladida, Platyhelminthes).

Using immunocytochemistry, serotonergic nerve elements were documented in the nervous system of the planarian Girardia tigrina. Serotonin-immunopositive components were observed in the brain, ventral, dorsal and longitudinal nerve cords, transverse nerve commissures connecting the nerve cords, and in the nerve plexus. Whole-mount preparations of G. tigrina were analyzed by fluorescent and confocal laser scanning microscopy. An essential quantitative morphometric measurement of serotonin-immunopositive structures was conducted in three body regions (anterior, middle, and posterior) of the planarian. The number of serotonin neurons was maximal in the head region. The ventral nerve cords gradually decreased in thickness from anterior to posterior body ends. Physiological action of exogenously applied serotonin was studied in G. tigrina for the first time. It was found that serotonin (0.1 and 1 µmol L-1) accelerated eye regeneration. The transcriptome sequencing performed for the first time for the planarian G. tigrina revealed the transcripts of the tryptophan hydroxylase (trph), amino acid decarboxylase (aadc) and serotonin transporter (sert) genes. The data obtained indicate the presence of the components of serotonin pathway in G. tigrina. The identified transcripts can take part in serotonin turnover and participate in the realization of biological effects of serotonin in planarians, associated with eyes regeneration and differentiation.

Active-Site Oxygen Accessibility and Catalytic Loop Dynamics of Plant Aromatic Amino Acid Decarboxylases from Molecular Simulations.

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.

Computer-aided design and implementation of efficient biosynthetic pathways to produce high added-value products derived from tyrosine in Escherichia coli.

Developing efficient bioprocesses requires selecting the best biosynthetic pathways, which can be challenging and time-consuming due to the vast amount of data available in databases and literature. The extension of the shikimate pathway for the biosynthesis of commercially attractive molecules often involves promiscuous enzymes or lacks well-established routes. To address these challenges, we developed a computational workflow integrating enumeration/retrosynthesis algorithms, a toolbox for pathway analysis, enzyme selection tools, and a gene discovery pipeline, supported by manual curation and literature review. Our focus has been on implementing biosynthetic pathways for tyrosine-derived compounds, specifically L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine, with significant applications in health and nutrition. We selected one pathway to produce L-DOPA and two different pathways for dopamine-one already described in the literature and a novel pathway. Our goal was either to identify the most suitable gene candidates for expression in Escherichia coli for the known pathways or to discover innovative pathways. Although not all implemented pathways resulted in the accumulation of target compounds, in our shake-flask experiments we achieved a maximum L-DOPA titer of 0.71g/L and dopamine titers of 0.29 and 0.21g/L for known and novel pathways, respectively. In the case of L-DOPA, we utilized, for the first time, a mutant version of tyrosinase from Ralstonia solanacearum. Production of dopamine via the known biosynthesis route was accomplished by coupling the L-DOPA pathway with the expression of DOPA decarboxylase from Pseudomonas putida, resulting in a unique biosynthetic pathway never reported in literature before. In the context of the novel pathway, dopamine was produced using tyramine as the intermediate compound. To achieve this, tyrosine was initially converted into tyramine by expressing TDC from Levilactobacillus brevis, which, in turn, was converted into dopamine through the action of the enzyme encoded by ppoMP from Mucuna pruriens. This marks the first time that an alternative biosynthetic pathway for dopamine has been validated in microbes. These findings underscore the effectiveness of our computational workflow in facilitating pathway enumeration and selection, offering the potential to uncover novel biosynthetic routes, thus paving the way for other target compounds of biotechnological interest.