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Mammalian Dopa decarboxylase: structure, catalytic activity and inhibition
Abstract
Mammalian Dopa decarboxylase catalyzes the conversion of L-Dopa and L-5-hydroxytryptophan to dopamine and serotonin, respectively. Both of them are biologically active neurotransmitters whose levels should be finely tuned. In fact, an altered concentration of dopamine is the cause of neurodegenerative diseases, such as Parkinson's disease. The chemistry of the enzyme is based on the features of its coenzyme pyridoxal 5'-phosphate (PLP). The cofactor is highly reactive and able to perform multiple reactions, besides decarboxylation, such as oxidative deamination, half-transamination and Pictet-Spengler cyclization. The structure resolution shows that the enzyme has a dimeric arrangement and provides a molecular basis to identify the residues involved in each catalytic activity. This information has been combined with kinetic studies under steady-state and pre-steady-state conditions as a function of pH to shed light on residues important for catalysis. A great effort in DDC research is devoted to design efficient and specific inhibitors in addition to those already used in therapy that are not highly specific and are responsible for the side effects exerted by clinical approach to either Parkinson's disease or aromatic amino acid decarboxylase deficiency.
... Kynurenine aminotransferase catalyzes the degradation of tryptophan to kynurenine catabolic intermediates, such as, kynurenic acid, 3-hydroxykynurenine, and quinolinic acid, which are neuroactive and related to human neurodegenerative disorders (20,21), making kynurenine aminotransferase a valid drug target. The major human neurotransmitters dopamine and serotonin are generated from L-DOPA and L-5-HTP, respectively, by L-3,4-dihydroxyphenylalanine (DOPA) decarboxylase, a PLP-dependent enzyme (22). DOPA decarboxylase degrades L-DOPA within the peripheral nervous system, making it a target in the management of Parkinson's disease (23). ...
... Usually, a nearby residue (including the previously PLP-linked catalytic Lys), which is a catalytic base, subtracts a proton from the resulting PLP-linked substrate, changing it into a carbanion intermediate. However, carbanion formation is also possible by the decarboxylation of the external aldimine substrate (22) or the attack of another cofactor like tetrahydrofolate (40). The resulting non-bonded electrons of the carbanion intermediate are stabilized by the elaborated electron movements through the extended conjugated π-bond system of PLP and the bound substrate via the quinonoid structure. ...
... For example, in GABA aminotransferase the active site Lys deprotonates the Cγ atom of GABA in the external aldimine structure, which is stabilized by the extended conjugated π-bond system and eventually leads to amino group transfer (15). In DOPA decarboxylase, the Cα decarboxylation generates a carbanion intermediate, which is also stabilized by the extended conjugated π-bond system of PLP (22). Alanine racemase has a different catalytic base on the opposite side of the typical catalytic Lys, based on the central PLP plane. ...
Pyridoxal 5'-phosphate (PLP)-dependent enzymes are ubiquitous, catalyzing various biochemical reactions of approximately 4% of all classified enzymatic activities. They transform amines and amino acids into important metabolites or signaling molecules and are important drug targets in many diseases. In the crystal structures of PLP-dependent enzymes, organic cofactor PLP showed diverse conformations depending on the catalytic step. The conformational change of PLP is essential in the catalytic mechanism. In the study, we review the sophisticated catalytic mechanism of PLP, especially in transaldimination reactions. Most drugs targeting PLP-dependent enzymes make a covalent bond to PLP with the transaldimination reaction. A detailed understanding of organic cofactor PLP will help develop a new drug against PLP-dependent enzymes.
... The versatility of PLP as a cofactor capable of mediating diverse reactions leads AADCs to be susceptible to undesirable off-pathway reactions (Fig. 7). First, as aforementioned, AADCs have been reported to carry out decarboxylation-dependent transamination as a side reaction (Bertoldi 2014;Bertoldi and Voltattorni 2003). Owing to the carbanionic character of C4′ as well as Cα in the quinonoid intermediate, protonation of C4′ could occur and result in a ketimine intermediate of which hydrolysis leads to a transamination pathway (Bertoldi 2014;Bertoldi and Voltattorni 2003). ...
... First, as aforementioned, AADCs have been reported to carry out decarboxylation-dependent transamination as a side reaction (Bertoldi 2014;Bertoldi and Voltattorni 2003). Owing to the carbanionic character of C4′ as well as Cα in the quinonoid intermediate, protonation of C4′ could occur and result in a ketimine intermediate of which hydrolysis leads to a transamination pathway (Bertoldi 2014;Bertoldi and Voltattorni 2003). The transamination leads to the conversion of the PLP cofactor into pyridoxamine 5′-phosphate (PMP), resulting in a gradual accumulation of the PMP form of AADCs incapable of initiating the decarboxylation reaction (Bertoldi andVoltattorni 2000, 2003). ...
... Owing to the carbanionic character of C4′ as well as Cα in the quinonoid intermediate, protonation of C4′ could occur and result in a ketimine intermediate of which hydrolysis leads to a transamination pathway (Bertoldi 2014;Bertoldi and Voltattorni 2003). The transamination leads to the conversion of the PLP cofactor into pyridoxamine 5′-phosphate (PMP), resulting in a gradual accumulation of the PMP form of AADCs incapable of initiating the decarboxylation reaction (Bertoldi andVoltattorni 2000, 2003). However, AADCs are known to suppress the undesirable C4′ protonation Fig. 5 Proposed reaction mechanism of AADCs. ...
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.
... The crystallographic data are summarized in Table 1. The structures display the characteristic architecture of the fold-type I superfamily, and as previously described, comprises of three domains, an N-terminal domain (residues 1-30), a PLP-binding large domain (residues 30-302) and a small C-terminal domain (residues 303-395) [23]. Residues (269/270-278) of the catalytic loop (CL), and residues 364-366 of a C-terminal loop were disordered and not modelled in all the structures (Figure 2a). ...
... The final step in the reaction is the poorly-described quinonoid-Cα protonation [9,10,23,24,25]. ...
... Protonation of QND-Cα happens on the re face of the intermediate that is solvent-exposed in both K245A-intermediate complexes, deterring the direct identification of the residue required for this function. Previous biochemical and computational studies on homologs indicate an indispensable catalytic role for the highly conserved Tyr273′ residue present in the CL of the adjacent subunit [9,18,23,24,25]. The composition and length (13-24 residues) of the CL region are divergent across orthologs. ...
PLP-dependent enzymes catalyze a plethora of chemical reactions affecting diverse physiological functions. Here we report the structural determinants of the reaction mechanism in a Group II PLP-dependent decarboxylase by assigning two early intermediates. The in-crystallo complexes of the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observation of the active site interactions. The structures reveal that a subtle rearrangement of a conserved Arg residue in concert with a water-mediated interaction with the carboxylate of the Dunathan intermediate, appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid. Modeling indicates that the conformational change of a dynamic catalytic loop to a closed form controls a conserved network of hydrogen bond interactions between catalytic residues to protonate the quinonoid. Our results provide a structural framework to elucidate mechanistic roles of residues that govern reaction specificity and catalysis in PLP-dependent decarboxylation.
... The genotype UAS-Sph1/+; tph-GAL4/+ used the tryptophan hydroxylase (tph-GAL4) GAL4 driver to express Sph-1 in serotonergic neurons, while the genotype UAS-Sph1/+; ddc-GAL4/+ used the Dopa-decarboxylase GAL4 driver (ddc-GAL4) to express Sph-1 in both dopaminergic and serotonergic neurons. This GAL4 driver has an enhancer of the Ddc gene, which drives expression in both types of aminergic neurons [18]. The genotype UAS-Sph1/+; th-GAL4 used the tyrosine hydroxylase driver (th-GAL4) to express Sph-1 only in dopaminergic neurons. ...
... In this study, we used the tryptophan hydroxylase driver (UAS-Sph1/+; tph-GAL4/+) to express Sph-1 in serotonergic neurons and the Dopa-decarboxylase driver (UAS-Sph1/+; ddc-GAL4/+) to express Sph-1 in both dopaminergic and serotonergic neurons simultaneously [18]. Our results show that lifespan is not affected when Sph-1 is expressed in the serotonergic system, although nicotine induced a small but significant increase in survival in these flies. ...
Synphilin-1 is a protein encoded by the human SNCAIP gene whose function has yet to be fully understood. However, it has been linked to familial Parkinson’s disease (PD). Synphilin-1 is a major component of the Lewy bodies found in neurons in the substantia nigra pars compacta of PD patients. Synphilin-1 expression in serotonergic and/or dopaminergic neurons of Drosophila melanogaster induces neurodegeneration, as well as motor and non-motor PD like symptoms. In this work, we examined the contribution of the serotonergic and dopaminergic circuits in the development of PD-like phenotypes. We found that olfactory and visual symptoms are majorly contributed by the serotonergic system, and that motor symptoms and reduction in survival are mainly contributed by the dopaminergic system. Chronic nicotine treatment was able to suppress several of these symptoms. These results indicate that both the serotonergic and dopaminergic systems contribute to different aspects of PD symptomatology and that nicotine has beneficial effects on specific symptoms.
... Concerning the latter, we aimed to elucidate the mechanism(s) mediating the down-regulation of DDC protein by HCV infection [7]. This includes the reduction of its total intracellular levels, detected in IF, as well as an accumulation of the~50 kDa DDC monomer with a concomitant reduction of a~120 kDa DDC immunoreactive SDS-resistant protein, which is possibly the dimeric catalytically active form of the protein or a yet unknown DDC isoform species [7,8]. As the subgenomic HCV JFH1 replicon, which expresses only the non-structural viral proteins, exerts the same effect on DDC as the full-length virus [7], we examined the role of individual non-structural proteins in modifying DDC levels. ...
... Concerning DDC, we have previously shown that HCV mediates an accumulation of the~50 kDa DDC monomer with a concomitant reduction of a~120 kDa DDC complex, possibly corresponding to the dimeric catalytically active form of the protein [7,8]. Moreover, HCV was observed to reduce the total DDC intracellular levels in IF [7]. ...
A bidirectional negative relationship between Hepatitis C virus (HCV) replication and gene expression of the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) was previously shown in the liver and attributed at least to an association of DDC with phosphatidylinositol 3-kinase (PI3K). Here, we report that the biosynthesis and uptake of catecholamines restrict HCV replication in hepatocytes, while HCV has developed ways to reduce catecholamine production. By employing gene silencing, chemical inhibition or induction of the catecholamine biosynthetic and metabolic enzymes and transporters, and by applying the substrates or the products of the respective enzymes, we unravel the role of the different steps of the pathway in viral infection. We also provide evidence that the effect of catecholamines on HCV is strongly related with oxidative stress that is generated by their autoxidation in the cytosol, while antioxidants or treatments that lower cytosolic catecholamine levels positively affect the virus. To counteract the effect of catecholamines, HCV, apart from the already reported effects on DDC, causes the down-regulation of tyrosine hydroxylase that encodes the rate-limiting enzyme of catecholamine biosynthesis and suppresses dopamine beta-hydroxylase mRNA and protein amounts, while increasing the catecholamine degradation enzyme monoamine oxidase. Moreover, the NS4B viral protein is implicated in the effect of HCV on the ratio of the ~50 kDa DDC monomer and a ~120 kDa DDC complex, while the NS5A protein has a negative effect on total DDC protein levels.
... PLPdependent decarboxylation occurs when, upon substrate binding, the carboxyl group is released as CO2 producing a quinonoid intermediate. The latter is then reprotonated at Cα, generating the amine product [70] (Figure 3D). Most of the biochemical features known on AADC until now are related to the enzyme from pig kidney, which shares 90% of sequence identity with the human counterpart. ...
... Although microbial and host AADC seem to share most of their biochemical features, their substrate preferences and physiological role seem different. Human AADC is the enzyme responsible for dopamine and serotonin biosynthesis, starting from the corresponding amino acids L-dopa and 5-hydroxyTrp, respectively [70]. AADC is expressed in both neuronal and non-neuronal tissues, with the latter being found mainly in the kidneys and in the gastrointestinal tract [80]. ...
The chemical processes taking place in humans intersects the myriad of metabolic pathways occurring in commensal microorganisms that colonize the body to generate a complex biochemical network that regulates multiple aspects of human life. The role of tryptophan (Trp) metabolism at the intersection between the host and microbes is increasingly being recognized, and multiple pathways of Trp utilization in either direction have been identified with the production of a wide range of bioactive products. It comes that a dysregulation of Trp metabolism in either the host or the microbes may unbalance the production of metabolites with potential pathological consequences. The ability to redirect the Trp flux to restore a homeostatic production of Trp metabolites may represent a valid therapeutic strategy for a variety of pathological conditions, but identifying metabolic checkpoints that could be exploited to manipulate the Trp metabolic network is still an unmet need. In this review, we put forward the hypothesis that pyridoxal 5′-phosphate (PLP)-dependent enzymes, which regulate multiple pathways of Trp metabolism in both the host and in microbes, might represent critical nodes and that modulating the levels of vitamin B6, from which PLP is derived, might represent a metabolic checkpoint to re-orienteer Trp flux for therapeutic purposes.
... However, 24 h after the second treatment, the movement speed of the rotenone-treated silkworms was significantly slower than that of the water-or DMSO-treated silkworms, suggesting that rotenone may induce motor dysfunction in the silkworms (Figure 10A). Dopamine is a neurotransmitter that regulates motion (Bertoldi, 2014). To investigate whether rotenone also affected dopamine synthesis, we analyzed the expression of Tyrosine hydroxylase (TH). ...
Parkinson’s disease (PD), ranking as the second most prevalent neurodegenerative disorder globally, presents a pressing need for innovative animal models to deepen our understanding of its pathophysiology and explore potential therapeutic interventions. The development of such animal models plays a pivotal role in unraveling the complexities of PD and investigating promising treatment avenues. In this study, we employed transcriptome sequencing on BmN cells treated with 1 μg/ml rotenone, aiming to elucidate the underlying toxicological mechanisms. The investigation brought to light a significant reduction in mitochondrial membrane potential induced by rotenone, subsequently triggering mitophagy. Notably, the PTEN induced putative kinase 1 (PINK1)/Parkin pathway emerged as a key player in the cascade leading to rotenone-induced mitophagy. Furthermore, our exploration extended to silkworms exposed to 50 μg/ml rotenone, revealing distinctive motor dysfunction as well as inhibition of Tyrosine hydroxylase (TH) gene expression. These observed effects not only contribute valuable insights into the impact and intricate mechanisms of rotenone exposure on mitophagy but also provide robust scientific evidence supporting the utilization of rotenone in establishing a PD model in the silkworm. This comprehensive investigation not only enriches our understanding of the toxicological pathways triggered by rotenone but also highlights the potential of silkworms as a valuable model organism for PD research.
... Aromatic l-amino acid decarboxylase (AAAD), a key enzyme implicated in the conversion of ldopa to dopamine, is encoded by the DDC gene. As AAAD is critical for the metabolism of ldopa, polymorphisms in the DDC gene have been deemed to modify the expression of AAAD and consequently promote inter-individual variations in treatment response and clinical heterogeneity (Bertoldi 2014). Devos et al., conducted a study to investigate the motor response to l-dopa in PD patients as a function of the DDC gene promoter polymorphisms (rs921451 T > C polymorphism (DDC (T/C)) and rs3837091 AGAG del (DDC (AGAG/-))). ...
Parkinson’s disease (PD) is a relentlessly growing neurodegenerative disorder clinically manifested by rigidity, tremors, and dyskinesia ascribed to loss of nigrostriatal dopaminergic innervation. Conventional therapies focus on alleviating disease symptoms as far as possible, yet lack the ability to halt the progression of the neurodegenerative process. A major historic turning point in PD therapy came through the inception of levodopa (l-dopa), however, its prolonged use is associated with complications and a decline in response. Some alternate modalities to treat PD are also available as dopamine agonists (DA), catechol-o-methyl transferase inhibitors (COMTIs), and non-dopaminergic drugs that could be used as a supportive therapy with l-dopa or alone. Nonetheless, these drugs are less effective than l-dopa in regulating motor symptoms. On the other hand, substantial inter-individual variations are observed in response to l-dopa. Although many factors can influence an individual’s response to therapy, a patient’s genetic makeup could be a starting point in creating precision medicine with greater safety and efficacy. Precision medicine that incorporates pharmacogenomics data can optimize patients’ response to PD drugs and facilitate treatment. In this review, we aim to present PD pathogenesis, challenges of current therapy, and pharmacogenetics aspects of levodopa.
... Aromatic amino acid decarboxylase (AADC) deficiency (OMIM #608643) is an autosomal recessive neurotransmitter disease caused by variants in the DDC gene (GRCh38.p13; chr7:50,447,733-50,576,163) encoding the AADC enzyme responsible for dopamine and serotonin synthesis from L-Dopa and L-5-hydroxytryptophan, respectively [1]. In the vast majority of cases, the disease onset is in the first months of life, and patients develop serious motor and neurodevelopmental symptoms causing severe disability. ...
A case of an adult with borderline AADC deficiency symptoms is presented here. Genetic analysis revealed that the patient carries two AADC variants (NM_000790.3: c.1040G > A and c.679G > C) in compound heterozygosis, resulting in p.Arg347Gln and p.Glu227Gln amino acid alterations. While p.Arg347Gln is a known pathogenic variant, p.Glu227Gln is unknown. Combining clinical features to bioinformatic and molecular characterization of the AADC protein population of the patient (p.Arg347Gln/p.Arg347Gln homodimer, p.Glu227Gln/p.Glu227Gln homodimer, and p.Glu227Gln/p.Arg347Gln heterodimer), we determined that: i) the p.Arg347Gln/p.Arg347Gln homodimer is inactive since the alteration affects a catalytically essential structural element at the active site, ii) the p.Glu227Gln/p.Glu227Gln homodimer is as active as the wild-type AADC since the alteration occurs at the surface and does not change the chemical nature of the amino acid, and iii) the p.Glu227Gln/p.Arg347Gln heterodimer has a catalytic efficiency 75% that of the wild-type since only one of the two active sites is compromised, thus demonstrating a positive complementation. By this approach, the molecular basis for the mild presentation of the disease is provided, and the experience made can also be useful for personalized therapeutic decisions in other mild AADC deficiency patients. Interestingly, in the last few years, many previously undiagnosed or misdiagnosed patients have been identified as mild cases of AADC deficiency, expanding the phenotype of this neurotransmitter disease.
... TH reaction is the first and rate-limiting step in the catecholamine biosynthetic pathway and is under tight short-term regulation by cytosolic catecholamine mediated feedback inhibition (especially DA) in conjunction with the phosphorylation/dephosphorylation of the regulatory domain Ser residues (40, 31, and 19) by a number of protein kinases/phosphatases (Dickson and Briggs, 2013). In the second step, cytosolic L-DOPA is effectively decarboxylated by a pyridoxal phosphate dependent enzymes, DOPA decarboxylase or non-specific aromatic amino acid decarboxylases (AADC) (Bertoldi, 2014) to DA in the cytosol and, cytosolic DA is actively sequestered into the synaptic vesicles for transient storage through a H + coupled transmembrane antiporter, vesicular monoamine transporter-2 (VMAT2) (Scheme 2; Wimalasena, 2011). The intragranular H + ion gradient required for functioning of VMAT2 mediated active vesicular uptake of DA against a steep concentration gradient is generated by an inward H + translocating V-ATPase in the synaptic vesicle membrane (Wimalasena, 2011). ...
Parkinson’s disease (PD) is an age-related irreversible neurodegenerative disease which is characterized as a progressively worsening involuntary movement disorder caused by the loss of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc). Two main pathophysiological features of PD are the accumulation of inclusion bodies in the affected neurons and the predominant loss of neuromelanin-containing DA neurons in substantia nigra pars compacta (SNpc) and noradrenergic (NE) neurons in locus coeruleus (LC). The inclusion bodies contain misfolded and aggregated α-synuclein (α-Syn) fibrils known as Lewy bodies. The etiology and pathogenic mechanisms of PD are complex, multi-dimensional and associated with a combination of environmental, genetic, and other age-related factors. Although individual factors associated with the pathogenic mechanisms of PD have been widely investigated, an integration of the findings to a unified causative mechanism has not been envisioned. Here we propose an integrated mechanism for the degeneration of DA neurons in SNpc and NE neurons in LC in PD, based on their unique high metabolic activity coupled elevated energy demand, using currently available experimental data. The proposed hypothetical mechanism is primarily based on the unique high metabolic activity coupled elevated energy demand of these neurons. We reason that the high vulnerability of a selective group of DA neurons in SNpc and NE neurons in LC in PD could be due to the cellular energy modulations. Such cellular energy modulations could induce dysregulation of DA and NE metabolism and perturbation of the redox active metal homeostasis (especially copper and iron) in these neurons.
... Since our PD cohort is on treatment (mixture of L-Dopa/ Carbidopa), they were expected to have lower levels of PLP as it is the binding target for Carbidopa (Bertoldi, 2014). Our results showed that the levels of pyridoxamine were significantly higher in PD patients than in reference control. ...
Introduction: Parkinson’s disease (PD) is the most common motor neurodegenerative disease worldwide. Given the complexity of PD etiology and the different metabolic derangements correlated to the disease, metabolomics profiling of patients is a helpful tool to identify patho-mechanistic pathways for the disease development. Dopamine metabolism has been the target of several previous studies, of which some have reported lower phenylalanine and tyrosine levels in PD patients compared to controls.
Methods: In this study, we have collected plasma from 27 PD patients, 18 reference controls, and 8 high-risk controls to perform a metabolomic study using liquid chromatography-electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS).
Results: Our findings revealed higher intensities of trans-cinnamate, a phenylalanine metabolite, in patients compared to reference controls. Thus, we hypothesize that phenylalanine metabolism has been shifted to produce trans-cinnamate via L-phenylalanine ammonia lyase (PAL), instead of producing tyrosine, a dopamine precursor, via phenylalanine hydroxylase (PAH).
Discussion: Given that these metabolites are precursors to several other metabolic pathways, the intensities of many metabolites such as dopamine, norepinephrine, and 3-hydroxyanthranilic acid, which connects phenylalanine metabolism to that of tryptophan, have been altered. Consequently, and in respect to Metabolic Control Analysis (MCA) theory, the levels of tryptophan metabolites have also been altered. Some of these metabolites are tryptamine, melatonin, and nicotinamide. Thus, we assume that these alterations could contribute to the dopaminergic, adrenergic, and serotonergic neurodegeneration that happen in the disease.
... L-Dopa decarboxylase (DDC; EC 4.1.1.28) is a pyridoxine-5-phosphate-requiring enzyme, which decarboxylates L-3,4-dihydroxyphenylalanine (L-Dopa) and 5-hydroxytryptophan (5-HTP) converting them to dopamine (DA) and serotonin (5-HT), respectively [7]. DDC is additionally known as aromatic L-amino acid decarboxylase (AADC), since it is thought to decarboxylate other aromatic L-amino acids in mammalian tissues [8][9][10]. ...
Oxidative stress is known to influence mRNA levels, translation, and proteolysis. The importance of oxidative stress has been demonstrated in several human diseases, including neurodegenerative disorders. L-Dopa decarboxylase (DDC) is the enzyme that converts L-Dopa to dopamine (DA). In spite of a large number of studies, little is known about the biological significance of the enzyme under physiological and pathological conditions. Here, we investigated the relationship between DDC expression and oxidative stress in human neural and non-neural cells. Oxidative stress was induced by treatment with H2O2. Our data indicated that mRNA and protein expression of DDC was enhanced or remained stable under conditions of ROS induction, despite degradation of total RNA and increased cytotoxicity and apoptosis. Moreover, DDC silencing caused an increase in the H2O2-induced cytotoxicity. The current study suggests that DDC is involved in the mechanisms of oxidative stress.
... Future studies should experimentally determine MAO-B substrate binding affinity when complexed with various spike protein variants, both biochemically and in the context of living systems. Alterations to monoamine metabolism with SARS-CoV-2 infection may also partly be amplified by the downregulation of L-DOPA decarboxylase (DDC) (Mpekoulis et al., 2021), which catalyzes L-DOPA to dopamine and, to a lesser degree, l-5-hydroxytryptophan to serotonin (Bertoldi, 2014). Altered monoamine metabolism may be of particular importance in severe cases of SARS-CoV-2 infection, as high plasma concentrations of homovanillic acid, an end-stage product of catechol-O-methyltransferase and monoamine oxidase metabolism, during infection was identified as a predictor of mortality (Richard et al., 2022). ...
SARS-CoV-2 infection is associated with both acute and post-acute neurological symptoms. Emerging evidence suggests that SARS-CoV-2 can alter mitochondrial metabolism, suggesting that changes in brain metabolism may contribute to the development of acute and post-acute neurological complications. Monoamine oxidase B (MAO-B) is a flavoenzyme located on the outer mitochondrial membrane that catalyzes the oxidative deamination of monoamine neurotransmitters. Computational analyses have revealed high similarity between the SARS-CoV-2 spike glycoprotein receptor binding domain on the ACE2 receptor and MAO-B, leading to the hypothesis that SARS-CoV-2 spike glycoprotein may alter neurotransmitter metabolism by interacting with MAO-B. Our results empirically establish that the SARS-CoV-2 spike glycoprotein interacts with MAO-B, leading to increased MAO-B activity in SH-SY5Y neuron-like cells. Common to neurodegenerative disease pathophysiological mechanisms, we also demonstrate that the spike glycoprotein impairs mitochondrial bioenergetics, induces oxidative stress, and perturbs the degradation of depolarized aberrant mitochondria through mitophagy. Our findings also demonstrate that SH-SY5Y neuron-like cells expressing the SARS-CoV-2 spike protein were more susceptible to MPTP-induced necrosis, likely necroptosis. Together, these results reveal novel mechanisms that may contribute to SARS-CoV-2-induced neurodegeneration.
... Moreover, in agreement with our results, the CSF DDC levels are not associated with clinical motor scores. An insufficient DDC synthesis leads to impaired motor coordination, disturbance in cognitive and physiological homeostasis, many neuropsychiatric disorders and severe developmental delay [46]. We observed correlations of DDC level with LEDD and LDD in PD patients; however, DDC was also significantly increased in drug-naïve patients. ...
Background
There is a need for biomarkers to support an accurate diagnosis of Parkinson’s disease (PD). Cerebrospinal fluid (CSF) has been a successful biofluid for finding neurodegenerative biomarkers, and modern highly sensitive multiplexing methods offer the possibility to perform discovery studies. Using a large-scale multiplex proximity extension assay (PEA) approach, we aimed to discover novel diagnostic protein biomarkers allowing accurate discrimination of PD from both controls and atypical Parkinsonian disorders (APD).
Methods
CSF from patients with PD, corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), multiple system atrophy and controls, were analysed with Olink PEA panels. Three cohorts were used in this study, comprising 192, 88 and 36 cases, respectively. All samples were run on the Cardiovascular II, Oncology II and Metabolism PEA panels.
Results
Our analysis revealed that 26 and 39 proteins were differentially expressed in the CSF of test and validation PD cohorts, respectively, compared to controls. Among them, 6 proteins were changed in both cohorts. Midkine (MK) was increased in PD with the strongest effect size and results were validated with ELISA. Another most increased protein in PD, DOPA decarboxylase (DDC), which catalyses the decarboxylation of DOPA ( L -3,4-dihydroxyphenylalanine) to dopamine, was strongly correlated with dopaminergic treatment. Moreover, Kallikrein 10 was specifically changed in APD compared with both PD and controls, but unchanged between PD and controls. Wnt inhibitory factor 1 was consistently downregulated in CBS and PSP patients in two independent cohorts.
Conclusions
Using the large-scale PEA approach, we have identified potential novel PD diagnostic biomarkers, most notably MK and DDC, in the CSF of PD patients.
... Dopamine biosynthesis has phenylalanine and tyrosine as precursors and uses the rate-limiting cofactors vitamin B3, iron (Fe), folate, tetrahydrobiopterin (BH4), vitamin D, and vitamin B6 to complete its synthesis. The synthesis of epinephrine from dopamine requires the cofactors vitamin C, copper (Cu), folate, S-adenosyl methionine (SAMe), and magnesium (Mg) [28,59,60] (Fig. 2). Serotonin (Fig. 3) is synthesized from tryptophan and requires the cofactors vitamins B3, vitamin B6, iron, folate, tetrahydrobiopterin (BH4), and vitamin D [61,62] (Fig. 3). ...
Background and purpose
The incidence of depression is increasing, despite continued advances in psychological and pharmacological interventions. New treatment approaches are urgently needed. Here we assess the effects on depression of individualized micronutrient supplementation, in concert with a standard set of lifestyle changes.
Methods
We conducted a small field-study with 17 participants in Austria. Patients with depression (n = 11) and healthy volunteers (n = 6) underwent laboratory serum analysis and filled out the DASS-21 and a questionnaire about their medical history and condition. The list of parameters to be tested in the serum analysis was derived from an expert heuristic compilation of factors known to influence depression, narrowed down to a workable list to be tested in this initial study. On the basis of the results, the participants (n = 17) received individualized recommendations for micronutrient supplementation, in collaboration with their treating physician. Participants followed the individual supplementation regime for two months, along with a standard set of lifestyle changes. After two months the laboratory serum analyses, the DASS-21, and the questionnaire were repeated.
Results
All patients with micronutrient deficiencies were in the patient group; none of the healthy volunteers showed any micronutrient deficiencies. After two months of precision supplementation and lifestyle changes, all but one patient had recovered from their depression or had considerably improved. The one patient who didn’t recover was the only one with a known trigger of their depression (trauma). Of 11 patients with depression, the trigger was unknown for the other ten.
Conclusions
These results have promising implications for further research, treatment, drug development, and public health. We propose that systematic screening of patients with symptoms of depression be developed for future research, medical care, and practice. Psychiatry and psychotherapy may see improved results once they no longer have to push against the underlying constraints of existing micronutrient deficiencies.
... The AADC enzyme is homodimeric, with each monomer consisting of a 309-residue large domain containing the pyridoxal 5′-phosphate (PLP) binding site, a C-terminal small domain of 86 residues, and an 85-residue N-terminal domain packed with the same elements of the neighboring subunit to stabilize the dimeric structure [9,10]. Biochemical and bioinformatics studies have revealed a variety of effects of pathogenic AADC variants, including structural and functional defects of the enzyme, such as decreased catalytic efficiency, decreased PLP binding affinity, and misfolding defects [11]. ...
... The AADC enzyme is homodimeric, with each monomer consisting of a 309-residue large domain containing the pyridoxal 5′-phosphate (PLP) binding site, a C-terminal small domain of 86 residues, and an 85-residue N-terminal domain packed with the same elements of the neighboring subunit to stabilize the dimeric structure [9,10]. Biochemical and bioinformatics studies have revealed a variety of effects of pathogenic AADC variants, including structural and functional defects of the enzyme, such as decreased catalytic efficiency, decreased PLP binding affinity, and misfolding defects [11]. ...
Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic.
... Two of these genes were annotated as DOPA decarboxylases (DDC or AADC; E.C.4.1.1.28), Smp_135230 and Smp_171580, enzymes that catalyze de carboxylation reactions of neurotransmitters and neuromodulators (De Luca et al., 2003;Bertoldi, 2014). For both genes, RNA-seq showed significantly higher transcript levels in bM compared with sM, and no transcripts in pairing-experienced (bisex females, bF) or pairing-unexperienced (single-sex females, sF) females (Lu et al., 2016;Lu et al., 2018). ...
Introduction
Schistosomes are the only mammalian flatworms that have evolved separate sexes. A key question of schistosome research is the male-dependent sexual maturation of the female since a constant pairing contact with a male is required for the onset of gonad development in the female. Although this phenomenon is long known, only recently a first peptide-based pheromone of males was identified that contributes to the control of female sexual development. Beyond this, our understanding of the molecular principles inducing the substantial developmental changes in a paired female is still rudimentary.
Objectives
Previous transcriptomic studies have consistently pointed to neuronal genes being differentially expressed and upregulated in paired males. These genes included Smp_135230 and Smp_171580, both annotated as aromatic-L-amino-acid decarboxylases (DOPA decarboxylases). Here, we characterized both genes and investigated their roles in male–female interaction of S. mansoni .
Methodologies/findings
Sequence analyses indicated that Smp_135230 represents an L-tyrosine decarboxylase (Sm tdc-1 ), whereas Smp_171580 represents a DOPA decarboxylase (Sm ddc-1 ). By qRT-PCR, we confirmed the male-specific and pairing-dependent expression of both genes with a significant bias toward paired males. RNA-interference experiments showed a strong influence of each gene on gonad differentiation in paired females, which was enhanced by double knockdown. Accordingly, egg production was significantly reduced. By confocal laser scanning microscopy, a failure of oocyte maturation was found in paired knockdown females. Whole-mount in situ hybridization patterns exhibited the tissue-specific occurrence of both genes in particular cells at the ventral surface of the male, the gynecophoral canal, which represents the physical interface of both genders. These cells probably belong to the predicted neuronal cluster 2 of S. mansoni.
Conclusion
Our results suggest that Sm tdc-1 and Sm ddc-2 are male-competence factors that are expressed in neuronal cells at the contact zone between the genders as a response of pairing to subsequently control processes of female sexual maturation.
... Antipsychotic efficacy may be linked to an efficacious reduction of dopamine synthesis capacity. Indeed, clinical studies have demonstrated that prolonged antipsychotic treatment reduces the activity of the DOPA decarboxylase enzyme [188], which is predicted to transform levodopa in dopamine [248]. Preclinical studies show that the inactivation of dopamine neurons is larger in animal models of hyperdopaminergia [78], thereby mimicking the most widely accepted pathophysiological mechanism of psychosis. ...
Treatment resistant schizophrenia (TRS) is characterized by a lack of, or suboptimal response to, antipsychotic agents. The biological underpinnings of this clinical condition are still scarcely understood. Since all antipsychotics block dopamine D2 receptors (D2R), dopamine-related mechanisms should be considered the main candidates in the neurobiology of antipsychotic non-response, although other neurotransmitter systems play a role. The aims of this review are: (i) to recapitulate and critically appraise the relevant literature on dopamine-related mechanisms of TRS; (ii) to discuss the methodological limitations of the studies so far conducted and delineate a theoretical framework on dopamine mechanisms of TRS; and (iii) to highlight future perspectives of research and unmet needs. Dopamine-related neurobiological mechanisms of TRS may be multiple and putatively subdivided into three biological points: (1) D2R-related, including increased D2R levels; increased density of D2Rs in the high-affinity state; aberrant D2R dimer or heteromer formation; imbalance between D2R short and long variants; extrastriatal D2Rs; (2) presynaptic dopamine, including low or normal dopamine synthesis and/or release compared to responder patients; and (3) exaggerated postsynaptic D2R-mediated neurotransmission. Future points to be addressed are: (i) a more neurobiologically-oriented phenotypic categorization of TRS; (ii) implementation of neurobiological studies by directly comparing treatment resistant vs. treatment responder patients; (iii) development of a reliable animal model of non-response to antipsychotics.
... It is a homodimeric αdecarboxylase that was first identified in 1938 in the kidney as essential for epinephrine synthesis, a product of dopamine (Hornykiewicz 2002). It catalyses the decarboxylation of L-DOPA and 5-HTP to give rise to dopamine and serotonin, respectively, and requires pyridoxal 5'-phosphate as cofactor (Bertoldi 2014). AADC is present in CNS and the peripheral tissues therefore, inhibitors of AADC, such as carbidopa, are used as adjunctive therapies in PD. ...
Parkinson’s disease (PD) is the second most common neurodegenerative disorder. The exact molecular mechanism of disease remains unclear. Several factors are proposed to play part including, but not limited to, decreased activity of mitochondrial complex I and lysosomal glucocerebrosidase enzymes and disrupted cellular antioxidant defence and lysosomal acidification. In addition, there is growing support for a role of organelle crosstalk between mitochondria and lysosome, the disruption of which is proposed to play part in PD pathology. The nature and consequence of this crosstalk remains unclear. The SH-SY5Y neuronal cell line model is commonly used to investigate PD mechanisms and potential therapeutics. However, functional analysis of the suitability of the cell line in its proliferative state or the necessity for differentiation remains unclear. Furthermore, iPSC-derived dopaminergic neurons are another commonly used model for PD and related diseases however, validating their functional dopamine metabolism is important to determine disease mechanism and test potential therapeutics. In this thesis, a host of biochemical tools, including HPLC measurement of neurotransmitter metabolites and enzyme activity assays, were used to elucidate the aforementioned ambiguities. The findings demonstrate that although there are similarities between proliferative and differentiated phenotypes of SH-SY5Y cells, there are also significant differences. Notably, the rate of dopamine turnover and the activity of lysosomal glucocerebrosidase were significantly higher in differentiated SH-SY5Y cells. In contrast, mitochondrial electron transport chain complexes’ activities were similar between the two phenotypes, despite a significant difference in mitochondrial content. Therefore, care should be taken when choosing either phenotype as a PD model. In addition, 4the findings demonstrate that inhibition of either mitochondrial complex I or lysosomal glucocerebrosidase affect both the ratio of pro-cathepsin D/cathepsin D protein expression and enzyme activity. Cathepsin D is one of the most ubiquitous lysosomal enzymes, the state of which can be used as reflection of the degree of lysosomal acidification. This shines a light on the potential involvement of both lysosomal glucocerebrosidase and mitochondrial complex I in maintenance of lysosomal acidification. This could be a consequence of a more dynamic crosstalk between mitochondria and lysosomes than previously thought. Moreover, the work presented provides a method for validation of the dysfunctional dopamine metabolism in iPSC derived dopaminergic neuronal disease models for aromatic amino acid decarboxylase deficiency and PD patients carrying mutations in PINK1. In addition, it provides a proof of concept for the effectiveness of both lentivirus-based gene therapy and levodopa treatment to restore dopamine metabolism in aromatic amino acid decarboxylase deficiency.
... These studies, however, although suggestive of complex formation prior to the transfer of PLP from the donor enzymes to the acceptor enzymes, do not address on an atomic level how the salvage and PLP-dependent enzymes recognize and interact with each other. DDC (or AADC) catalyzes the conversion of aromatic amino acids to their corresponding amines during the synthesis of a variety of essential neurotransmitters, e.g., decarboxylation of L-DOPA into dopamine (DA), 5-HTP to serotonin, L-tyrosine to tyramine, and tryptophan to tryptamine [17,18]. As a result of its essential involvement in neurotransmitter biosynthesis, DDC has become a major target for research on Parkinson's disease, depression, and other neurological illnesses [18][19][20][21][22][23][24]. ...
Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5′-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.
... Dopamine biosynthesis has phenylalanine and tyrosine as precursors and uses the rate-limiting cofactors vitamin B3, iron (Fe), tetrahydrobiopterin (BH4), vitamin D, and vitamin B6 to complete its synthesis. The synthesis of epinephrine from dopamine requires the cofactors vitamin C, copper (Cu), S-adenosyl methionine (SAMe), and magnesium (Mg) [12] [13] [14] (Fig. 1). ...
Background and purpose: The incidence of depression is increasing, despite continued advances in psychological and pharmacological interventions. New treatment approaches are urgently needed. Here we assess the effects on depression of individualized micronutrient supplementation, in concert with a standard set of lifestyle changes.
Methods: We conducted a small field-study with 17 participants in Austria. Patients with depression (n = 11) and healthy volunteers (n = 6) underwent laboratory serum analysis and filled out the DASS-21 and a questionnaire about their medical history and condition. The list of parameters to be tested in the serum analysis was derived from an expert heuristic compilation of factors known to influence depression, narrowed down to a workable list to be tested in this initial study. On the basis of the results, the participants (n = 17) received individualized recommendations for micronutrient supplementation, in collaboration with their treating physician. Participants followed the individual supplementation regime for two months, along with a standard set of lifestyle changes. After two months the laboratory serum analyses, the DASS-21, and the questionnaire were repeated.
Results: All patients with micronutrient deficiencies were in the patient group; none of the healthy volunteers showed any micronutrient deficiencies. After two months of precision supplementation and lifestyle changes, all but one patient had recovered from their depression or had considerably improved. The one patient who didn’t recover was the only one with a known trigger of their depression (trauma). Of 11 patients with depression, the trigger was unknown for the other ten.
Conclusions: These results have promising implications for further research, treatment, drug development, and public health. We propose that systematic screening of patients with symptoms of depression be developed for future research, medical care, and practice. Psychiatry and psychotherapy may see improved results once they no longer have to push against the underlying constraints of existing micronutrient deficiencies.
... Expression and puri cation of dopa decarboxylase HaDDC and its catalytic speci city towards L-trp and 5-HTP Dopa decarboxylase from Drosophila melanogaster (drDDC) has attracted signi cant attention, due to its activity for both 5-HTP and L-DOPA but with no activity to L-trp [19][20][21]. In this study, we used the DDC from Harmonia axyridis (HaDDC, GenBank: AMQ 13055.1) ...
Background: L-Tryptophan (L-trp) derivatives such as 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT), N-Acetyl-5-hydroxytryptamine and melatonin are important molecules with pharmaceutical interest. Among, 5-HT is an inhibitory neurotransmitter with proven benefits for treating the symptoms of depression. At present, 5-HT depends on plant extraction and chemical synthesis, which limits its mass production and causes environmental problems. Therefore, it is necessary to develop an efficient, green and sustainable biosynthesis method to produce 5-HT.
Results: Here we propose a one-pot production of 5-HT from L-trp via two enzyme cascades for the first time. First, a chassis cell that can convert L-trp into 5-HTP was constructed by heterologous expression of tryptophan hydroxylase from Schistosoma mansoni (SmTPH) and an artificial endogenous BH4 module. Then, dopa decarboxylase from Harminia axyridis (HaDDC), which can specifically catalyse 5-HTP to 5-HT, was used for 5-HT production. The cell factory, E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH, which contains SmTPH and HaDDC, was constructed for 5-HT synthesis. The highest concentration of 5-HT reached 414.5 ± 1.6 mg/L (with conversion rate of 43 mol%) at the optimal conditions (induced temperature, 25℃; IPTG concentration, 0.5 mM; catalysis temperature, 30℃; catalysis time, 72 h).
Conclusions: This protocol provided an efficient one-pot method for converting L-trp into 5-HT production, which opens up possibilities for the practical biosynthesis of natural 5-HT at an industrial scale.
... DDC acts as an endogenous modulator of central nerve transmission through its role in the biosynthesis of trace amines. 37 Therefore, DDC is a potential susceptibility gene for various neuropsychiatric disorders. Some studies have focused on the relationship between DDC and miRNAs. ...
Objective:
Bipolar I disorder (BD-I) is a type of bipolar spectrum disorder characterized by manic or mixed episodes. Detecting microRNA regulations as epigenetic actors in BD-I is important to elucidate the pathogenesis of the disease and reveal the potential of microRNAs (miRNAs) as biomarkers.
Methods:
We evaluated the expression profile of six candidate miRNAs (hsa-miR-145-5p, hsa-miR376a-3p, hsa-miR-3680-5p, hsa-miR-4253-5p, hsa-miR-4482-3p, and hsa-miR-4725) in patients with BD-I and in healthy controls (aged 11-50 years). We also determined the potential target genes of these miRNAs through in silico analysis. The diagnostic values of the miRNAs were calculated through receiver operating characteristic curve analysis.
Results:
Four miRNAs were upregulated (hsa-miR-376a-3p, hsa-miR-3680-5p, hsa-miR-4253-5p, hsa-miR-4482-3p) and hsa-miR-145-5p was downregulated in patients (p < 0.001). The target gene analyses showed that hsa-miR-145-5p specifically targets the dopamine decarboxylase (DDC) gene. The area under the curve of hsa-miR-145-5p was 0.987.
Conclusion:
: Differential expression of five miRNAs in peripheral blood may be associated with the pathogenesis of BD-I, and hsa-miR-145-5p has potential as a BD-I biomarker. This miRNA can be used in dopamine-serotonin regulation and dose adjustment in drug therapy via the DDC gene.
... Structurally, they all belong to the aspartate aminotransferase family (or Fold-Type I) [3,4] of PLPdependent enzymes and are functional asymmetric homodimers with the CL of each subunit exposed to the solvent and able to insert into the opposite active site in the so-called active closed enzymatic form (Supplemental Results). One of the best characterized among them is mammalian aromatic amino acid decarboxylase (AADC) responsible for the synthesis of dopamine and serotonin from l-3,4-dihydroxyphenylalanine (l-Dopa) and l-5-hydroxytryptophan (l-5HTP), respectively [5]. The spatial structure of pig hol-oAADC has been solved in the absence and in the presence of the inhibitor carbiDopa (cDopa) [6] that, although irreversibly bound to PLP through its hydrazine group, mimics the substrate binding mode, especially in the positioning of the catechol moiety. ...
Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C > T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5’-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy.
... Dopa decarboxylase from Drosophila melanogaster (drDDC) has attracted significant attention, due to its activity for both 5-HTP and l-DOPA but with no activity to l-Trp [19][20][21]. In this study, we used the DDC from Harmonia axyridis (HaDDC, GenBank: AMQ 13055.1) ...
Background
l-Tryptophan (l-Trp) derivatives such as 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT), N-Acetyl-5-hydroxytryptamine and melatonin are important molecules with pharmaceutical interest. Among, 5-HT is an inhibitory neurotransmitter with proven benefits for treating the symptoms of depression. At present, 5-HT depends on plant extraction and chemical synthesis, which limits its mass production and causes environmental problems. Therefore, it is necessary to develop an efficient, green and sustainable biosynthesis method to produce 5-HT.
Results
Here we propose a one-pot production of 5-HT from l-Trp via two enzyme cascades for the first time. First, a chassis cell that can convert l-Trp into 5-HTP was constructed by heterologous expression of tryptophan hydroxylase from Schistosoma mansoni (SmTPH) and an artificial endogenous tetrahydrobiopterin (BH4) module. Then, dopa decarboxylase from Harminia axyridis (HaDDC), which can specifically catalyse 5-HTP to 5-HT, was used for 5-HT production. The cell factory, E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH, which contains SmTPH and HaDDC, was constructed for 5-HT synthesis. The highest concentration of 5-HT reached 414.5 ± 1.6 mg/L (with conversion rate of 25.9 mol%) at the optimal conditions (substrate concentration,2 g/L; induced temperature, 25℃; IPTG concentration, 0.5 mM; catalysis temperature, 30℃; catalysis time, 72 h).
Conclusions
This protocol provided an efficient one-pot method for converting. l-Trp into 5-HT production, which opens up possibilities for the practical biosynthesis of natural 5-HT at an industrial scale.
... Structurally, they all belong to the aspartate aminotransferase family (or Fold-Type I) [3,4] of PLP-dependent enzymes and are functional asymmetric homodimers with the CL of each subunit exposed to the solvent and able to insert into the opposite active site in the so-called active closed enzymatic form (Supplemental Results). One of the best characterized among them is mammalian aromatic amino acid decarboxylase (AADC) responsible for the synthesis of dopamine and serotonin from L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (L-5HTP), respectively [5]. The spatial structure of pig holoAADC has been solved in the absence and in the presence of the inhibitor carbiDopa (cDopa) [6] that, although irreversibly bound to PLP through its hydrazine group, mimics the substrate binding mode, especially in the positioning of the catechol moiety. ...
Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic disfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of the important neurotransmitters dopamine, norepinephrine, epinephrine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c989C>T (Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site crevice as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5’-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy.
... • decarboxylation of L-Dopa to dopamine, 5-hydroxytryptophan (5-HTP) to serotonin and also other aromatic acids to corresponding amines • supply organism with essential neurotransmitters • implication in Parkinson's disease [38] • AR coactivator • NED marker • modulator of AR-regulated genes [39][40][41] Class III β-tubulin (TUBBIII) ...
Neuroendocrine prostate cancer (NEPC) represents a variant of prostate cancer that occurs in response to treatment resistance or, to a much lesser extent, de novo. Unravelling the molecular mechanisms behind transdifferentiation of cancer cells to neuroendocrine-like cancer cells is essential for development of new treatment opportunities. This review focuses on summarizing the role of small molecules, predominantly microRNAs, in this phenomenon. A published literature search was performed to identify microRNAs, which are reported and experimentally validated to modulate neuroendocrine markers and/or regulators and to affect the complex neuroendocrine phenotype. Next, available patients’ expression datasets were surveyed to identify deregulated microRNAs, and their effect on NEPC and prostate cancer progression is summarized. Finally, possibilities of miRNA detection and quantification in body fluids of prostate cancer patients and their possible use as liquid biopsy in prostate cancer monitoring are discussed. All the addressed clinical and experimental contexts point to an association of NEPC with upregulation of miR-375 and downregulation of miR-34a and miR-19b-3p. Together, this review provides an overview of different roles of non-coding RNAs in the emergence of neuroendocrine prostate cancer.
... We take the multiplicative average of these rather different values and use k 6 = √ 0.028 · 4.27 mM = 346 µM. The turnover number of the enzyme was determined to be 5.1 s −1 or 306 min −1 [88]. Assuming (rather arbitrarily) a DCC concentration of 10 −8 M, we obtain a V max (k 5 ) value of 3.06 µM/min, which is used in the calculations. ...
Dopamine (DA) is an important signal mediator in the brain as well as in the periphery. The term “dopamine homeostasis” occasionally found in the literature refers to the fact that abnormal DA levels can be associated with a variety of neuropsychiatric disorders. An analysis of the negative feedback inhibition of tyrosine hydroxylase (TH) by DA indicates, with support from the experimental data, that the TH-DA negative feedback loop has developed to exhibit 3,4-dihydroxyphenylalanine (DOPA) homeostasis by using DA as a derepression regulator. DA levels generally decline when DOPA is removed, for example, by increased oxidative stress. Robust DOPA regulation by DA further implies that maximum vesicular DA levels are established, which appear necessary for a reliable translation of neural activity into a corresponding chemical transmitter signal. An uncontrolled continuous rise (windup) in DA occurs when Levodopa treatment exceeds a critical dose. Increased oxidative stress leads to the successive breakdown of DOPA homeostasis and to a corresponding reduction in DA levels. To keep DOPA regulation robust, the vesicular DA loading requires close to zero-order kinetics combined with a sufficiently high compensatory flux provided by TH. The protection of DOPA and DA due to a channeling complex is discussed
... Through regulating the synthesis of DA, one of the crucial neurotransmitters in the central nerve center, TH and DDC modulate mood and behavior (Volkow, Wise, & Baler, 2017). TH and DDC are involved in the catalysis of 5-HT and NE, which play crucial roles in depressive mood (Bertoldi, 2014;Greengard, 2001). ...
Electroacupuncture (EA) is used as an adjunctive treatment for depression. This study was conducted to evaluate the efficacy and mechanisms of EA in the depressive rat model induced by chronic unpredictable mild stress (CUMS) in male adult Wistar rats. The underlying mechanisms were explored by using isobaric tags for relative and absolute quantitation (iTRAQ)‐based proteomic analysis of the proteins in the prefrontal cortex (PFC), and observing the number of the PFC neurons stained with hematoxylin and eosin (H&E) and synaptic morphological changes under transmission electron microscopy (TEM). The results showed that EA plus paroxetine (EA + Par) for 1 week significantly relieved depression‐like anhedonia symptoms and improved anxiety‐like behavior, accompanied by the improvements in synaptic morphology and a significant increase of PFC neurons. Moreover, EA or paroxetine alone significantly alleviated anhedonia symptoms after 2 weeks of intervention. Additionally, iTRAQ analysis showed that dopaminergic signaling was significantly altered in CUMS rats after 1 week of EA treatment. As the critical enzyme of this pathway, aromatic‐l‐amino‐acid decarboxylase (DDC) was significantly upregulated after the treatment with EA + Par for 1 week. These findings suggested that the dopaminergic signaling pathway in PFC may be involved in the antidepressant mechanisms of EA.
... Recently, gene therapy for AADC has shown promise for improving the general condition of patients [9,20] However, early diagnosis and treatment are important to improve the outcomes and reduce the parental stress related to diagnostic delay. [1,9,20] Although it is well known that 3-OMD levels are elevated in patients with AADC deficiency [21,22] there are few data on the frequency of AADC deficiency worldwide. The relatively few reported cases are an indication that this condition is rare. ...
Objective
Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare inherited autosomal recessive disorder of biogenic amine metabolism. Diagnosis requires analysis of neurotransmitter metabolites in cerebrospinal fluid, AADC enzyme activity analysis, or molecular analysis of the DDC gene. 3-O-methyldopa (3-OMD) is a key screening biomarker for AADC deficiency.
Methods
We describe a rapid method for 3-OMD determination in dried blood spots (DBS) using flow-injection analysis tandem mass spectrometry with NeoBase™ 2 reagents and ¹³C6-tyrosine as an internal standard, which are routinely used in high-throughput newborn screening. We assessed variability using quality control samples over a range of 3-OMD concentrations.
Results
Within-day and between-day precision determined with quality control samples demonstrated coefficients of variation <15%. 3-OMD concentrations in 1000 healthy newborns revealed a mean of 1.33 μmol/L (SD ± 0.56, range 0.61–3.05 μmol/L), 100 non-AADC control subjects (age 7 days – 1 year) showed a mean of 1.19 μmol/L (SD ± 0.35–2.00 μmol/L), and 81 patients receiving oral L-Dopa had a mean 3-OMD concentration of 14.90 μmol/L (SD ± 14.18, range 0.4–80.3 μmol/L). A patient with confirmed AADC was retrospectively analyzed and correctly identified (3-OMD 10.51 μmol/L). In April 2020, we started a pilot project for identifying AADC deficiency in DBSs routinely submitted to the expanded newborn screening program. 3-OMD concentrations were measured in 21,867 samples; no patients with AADC deficiency were identified. One newborn had a high 3-OMD concentration due to maternal L-Dopa treatment.
Discussion
We demonstrated a rapid new method to identify AADC deficiency using reagents and equipment already widely used in newborn screening programs. Although our study is limited, introduction of our method in expanded neonatal screening is feasible and could facilitate deployment of screening, allowing for early diagnosis that is important for effective treatment.
... Both the phosphorylated alcohol and amine forms (pyridoxine phosphate and pyridoxamine phosphate) are converted to pyridoxal phosphate by pyridoxine phosphate oxidase (PNPO) [79]. Pyridoxal phosphate is a cofactor for enzymes catalyzing decarboxylase reactions in gamma-aminobutyric acid (GAD1, GAD2) [80] and serotonin/dopamine biosynthesis (DDC) [81]; as well as for enzymes catalyzing transamination reactions (e.g. GOT1, GOT2, GPT, GPT2) [82], cysteine synthesis (CTH) [83], heme synthesis (ALAS1, ALAS2) [84], carnitine synthesis (3-hydroxy-6-N-trimethyllysine aldolase, gene unidentified) [85], niacin synthesis (KYNU) [86], and sphingolipid synthesis (SPTLC1, SPTLC2) [87]. ...
The kinetics and localization of the reactions of metabolism are coordinated by the enzymes that catalyze them. These enzymes are controlled via a myriad of mechanisms including inhibition/activation by metabolites, compartmentalization, thermodynamics, and nutrient sensing-based transcriptional or post-translational regulation; all of which are influenced as a network by the activities of metabolic enzymes and have downstream potential to exert direct or indirect control over protein abundances. Considering many of these enzymes are active only when one or more vitamin cofactors are present; the availability of vitamin cofactors likely yields a systems-influence over tissue proteomes. Furthermore, vitamins may influence protein abundances as nuclear receptor agonists, antioxidants, substrates for post-translational modifications, molecular signal transducers, and regulators of electrolyte homeostasis. Herein, studies of vitamin intake are explored for their contribution to unraveling vitamin influence over protein expression. As a body of work, these studies establish vitamin intake as a regulator of protein abundance; with the most powerful demonstrations reporting regulation of proteins directly related to the vitamin of interest. However, as a whole, the field has not kept pace with advances in proteomic platforms and analytical methodologies, and has not moved to validate mechanisms of regulation or potential for clinical application.
... DDC, also known as aromatic amino acid decarboxylase (AADC), has a broad substrate specificity being able to convert both L-dopa and L-5-hydroxytryptophan to dopamine and serotonin (Fig. 3a), respectively, in addition to other aromatic amino acids, producing the so-called trace amines (Bertoldi 2014). A scheme of all reactive substrates or products of DDC is reported in Fig. 4. A decarboxylationdependent transamination was identified as a side reaction for DDC (O'Leary and Baughn 1977). ...
The versatility of reactions catalyzed by pyridoxal 5′-phosphate (PLP) enzymes is largely due to the chemistry of their extraordinary catalyst. PLP is necessary for many reactions involving amino acids. Reaction specificity is controlled by the orientation of the external aldimine intermediate that is formed upon addition of the amino acidic substrate to the coenzyme. The breakage of a specific bond of the external aldimine gives rise to a carbanionic intermediate. From this point, the different reaction pathways diverge leading to multiple activities: transamination, decarboxylation, racemization, elimination, and synthesis. A significant novelty appeared approximately 30 years ago when it was reported that some PLP-dependent decarboxylases are able to consume molecular oxygen transforming an amino acid into a carbonyl compound. These side paracatalytic reactions could be particularly relevant for human health, also considering that some of these enzymes are responsible for the synthesis of important neurotransmitters such as γ-aminobutyric acid, dopamine, and serotonin, whose dysregulation under oxidative conditions could have important implications in neurodegenerative states. However, the reactivity of PLP enzymes with dioxygen is not confined to mammals/animals. In fact, some plant PLP decarboxylases have been reported to catalyze oxidative reactions producing carbonyl compounds. Moreover, other recent reports revealed the existence of new oxidase activities catalyzed by new PLP enzymes, MppP, RohP, Ind4, CcbF, PvdN, Cap15, and CuaB. These PLP enzymes belong to the bacterial and fungal kingdoms and are present in organisms synthesizing bioactive compounds. These new PLP activities are not paracatalytic and could only scratch the surface on a wider and unexpected catalytic capability of PLP enzymes.
Genomic imprinting in the brain is theorized to provide parental control over offspring social behaviors. Noncanonical genomic imprinting is a form of epigenetic regulation in which one of a gene’s alleles, either that of maternal or paternal inheritance, exhibits a bias towards higher expression of one parental allele compared to the other. This bias can differ depending on tissue type, and the degree of the parental allele expression bias can even vary across anatomical domains within the same tissue. Dopa decarboxylase ( Ddc ) and tyrosine hydroxylase ( Th ) are both noncanonically imprinted genes that preferentially express their maternal alleles in the brain and Ddc also has a paternal allele expression bias in the periphery. These two genes encode catecholamine synthesis enzymes for the production of dopamine (DA), norepinephrine (NE), and epinephrine (E), and Ddc is also in the serotonin (5-HT) synthesis pathway. These four neurotransmitters are critical regulators of social behavior and disruptions to them are implicated in human mental illnesses. Here we investigated the functional effects of noncanonical imprinting of Ddc and Th on social behavior in mice. By using reciprocal heterozygous mutant mice, we tested the impacts of Ddc and/or Th maternally and paternally inherited alleles on aggression, social recognition, dominance, and social preference behaviors. We found that Ddc paternal-null alleles affect aggression and social recognition behavior, Th maternal-null alleles affect sociability preferences, and compound inheritance of Th and Ddc maternal-null alleles influence preferences for social novelty. These results are consistent with Th and Ddc maternal allele biased expression in central monoaminergic systems regulating sociability, and Ddc paternal allele biased expression in peripheral monoaminergic systems regulating aggression and social recognition.
5-Hydroxytryptamine (5-HT) and its derivative bufotenine, which possess important physiological functions, are the primary active components in the secretions of toad parotid and skin gland. However, the biosynthetic pathway of these substances remains unclear in toads. To characterize toad's Aromatic-L-amino-acid decarboxylase (AADC), the key enzyme in the predicted 5-HT derivatives biosynthetic pathway, the full-length cDNA of AADC from Bufo bufo gargarizans (BbgAADC) was cloned from the parotoid gland of B. bufo gargarizans. The recombinant BbgAADC exhibited optimal expression in E. coli BL21 (DE3) containing pCold-BbgAADC after induction for 16 h at 15 °C with 0.3 mM IPTG, resulting in substantial yields of soluble proteins. The enzymological properties of BbgAADC were assessed, and it was determined that the optimal reaction temperature was 37 °C, the optimal pH was 8.6, and the optimum molar ratio of pyridoxal-5′-phosphate (PLP) to BbgAADC was found to be 3.6:1. Additionally, high substrate specificity was observed, as BbgAADC could catalyze the production of 5-HT from 5-hydroxytryptophan (5-HTP) but not dopamine or tryptamine from levodopa or tryptophan, respectively. The Km of the recombinant protein BbgAADC was 0.2918 mM and the maximum reaction rate (Vmax) was 1.182 μM·min−1 when 5-HTP was used as substrate. The Kcat was 0.0545 min−1, and Kcat/Km was 0.1868 mM−1·min−1. To elucidate the mechanism of BbgAADC, molecular docking was performed with PLP and 5-HTP, or the external aldimine formed by 5-HTP and PLP. The results indicated that the active sites for BbgAADC to bind with PLP were K303, H192, N300, A148, F309, T246, A273, and T147. W71, Y79, F80, P81, T82, H192, T246, N300, H302, F309, and R477 served as catalytically active sites for the binding of BbgAADC to 5-HTP. Furthermore, R447, W71, S149, N300, A148, and T147 of BbgAADC were involved in the decarboxylation reaction of the aldimine formed by PLP and 5-HTP.
Camponotus floridanus ants show altered behaviors followed by a fatal summiting phenotype when infected with manipulating Ophiocordyceps camponoti-floridani fungi. Host summiting as a strategy to increase transmission is also observed with parasite taxa beyond fungi, including aquatic and terrestrial helminths and baculoviruses. The drastic phenotypic changes can sometimes reflect significant molecular changes in gene expression and metabolite concentrations measured in manipulated hosts. Nevertheless, the underlying mechanisms still need to be fully characterized. To investigate the small molecules producing summiting behavior, we infected C. floridanus ants with O. camponoti-floridani and sampled their heads for LC–MS/MS when we observed the characteristic summiting phenotype. We link this metabolomic data with our previous genomic and transcriptomic data to propose mechanisms that underlie manipulated summiting behavior in “zombie ants.” This “multiomic” evidence points toward the dysregulation of neurotransmitter levels and neuronal signaling. We propose that these processes are altered during infection and manipulation based on (1) differential expression of neurotransmitter synthesis and receptor genes, (2) altered abundance of metabolites and neurotransmitters (or their precursors) with known behavioral effects in ants and other insects, and (3) possible suppression of a connected immunity pathway. We additionally report signals for metabolic activity during manipulation related to primary metabolism, detoxification, and anti-stress protectants. Taken together, these findings suggest that host manipulation is likely a multi-faceted phenomenon, with key processes changing at multiple levels of molecular organization.
Aromatic amino acid decarboxylase is a pyridoxal 5'-phosphate-dependent enzyme responsible for the synthesis of the neurotransmitters, dopamine and serotonin. Here, by a combination of bioinformatic predictions and analyses, phosphorylation assays, spectroscopic investigations and activity measurements, we determined that Ser-193, a conserved residue located at the active site, can be phosphorylated, increasing catalytic efficiency. In order to determine the molecular basis for this functional improvement, we determined the structural and kinetic properties of the site-directed variants S193A, S193D and S193E. While S193A retains 27% of the catalytic efficiency of wild-type, the two acidic side chain variants are impaired in catalysis with efficiencies of about 0.15% with respect to the wild-type. Thus, even if located at the active site, Ser-193 is not essential for enzyme activity. We advance the idea that this residue is fundamental for the correct architecture of the active site in terms of network of interactions triggering catalysis. This role has been compared with the properties of the Ser-194 of the highly homologous enzyme histidine decarboxylase whose catalytic loop is visible in the spatial structure, allowing us to propose the validation for the effect of the phosphorylation. The effect could be interesting for AADC deficiency, a rare monogenic disease, whose broad clinical phenotype could be also related to post translational AADC modifications.
Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive neurometabolic disorder caused by biallelic pathogenic variants in the DDC gene and mainly characterized by developmental delay, hypotonia, and oculogyric crises. Early diagnosis is crucial for correct patient management; however, many patients remain misdiagnosed or undiagnosed due to the rarity and clinical heterogeneity of the disorder especially in the milder forms. Here, we applied exome sequencing approach by screening 2000 paediatric patients with neurodevelopmental disorders to identify possible new AADC variants and AADC deficiency patients. We identified five distinct DDC variants in two unrelated individuals. Patient #1 harboured two compound heterozygous DDC variants: c.436-12T > C and c.435 + 24A>C and presented with psychomotor delay, tonic spasms, and hyperreactivity. Patient #2 had three homozygous AADC variants: c.1385G > A; p.Arg462Gln, c.234C > T; p.Ala78 = , and c.201 + 37A > G and presented with developmental delay and myoclonic seizures. The variants were classified as benign class I variants and therefore non-causative according to the ACMG/AMP guidelines. Since the AADC protein is a structural and functional obligate homodimer, we evaluated the possible AADC polypeptide chain combinations in the two patients and determined the effects resulting from the amino acid substitution Arg462Gln. Our patients carrying DDC variants presented clinical manifestations not precisely overlapped to the classical symptoms exhibited by the most severe AADC deficiency cases. However, screening data derived from exome sequencing in patients featuring wide-range symptoms related to neurodevelopmental disorders may help to identify AADC deficiency patients, especially when applied to larger cohorts.
Parkinson's disease is a progressive neurodegenerative disease affecting more than 10 million patients worldwide. The leading cause of this pathological condition is an imbalance between dopaminergic and cholinergic systems due to dopaminergic neurons' degeneration in the nigrostriatal pathways. The primary goal of Parkinson's therapy is to correct the levels of the mentioned neurotransmitters, and the administration of Levodopa has been accepted as a "gold standard" treatment. The amino acid precursor can successfully control the symptoms by compensating for the reduced concentration of endogenous dopamine and activating postsynaptic D-receptors in the striatum.
The intensive enzymatic degradation of levodopa in the gastrointestinal tract is the main reason for its low concentration in the midbrain (~1%) and the increased frequency of adverse drug reactions. Despite numerous attempts to improve clinical efficacy, increasing bioavailability and reducing side effects remain difficult. This makes it necessary to use innovative drug delivery systems capable of overcoming the problems mentioned above.
This literature review presents new technological approaches for improved delivery of levodopa to the central nervous system. Nanoparticles, liposomes, cyclodextrin complexes, carbon nanotubes, and others represent promising platforms for the delivery and controlled release of the dopamine precursor. With the ability to ensure optimal bioavailability, constant plasma concentration, minimal peripheral degradation, and reduced adverse drug reactions, they successfully overcome the shortcomings of conventional levodopa-containing dosage forms.
Diverse natural product carbocycles and heterocycles are continuously of interest to the biochemical, synthetic, and medicinal communities. The biosynthetic pathways that lead to structurally diverse diterpenes, and other natural products, involve genes that are both highly conserved and mutable in their characteristic. These properties help ensure that healthy ecosystems continue generating novel complex molecules with diverse biological activity. Many synthetic approaches towards these natural products have been described, however, as known structural diversity in nature increases so does the need for novel methods to access the relevant structural analogues. The total synthesis of (±)-aspewentin A via a tandem Michael-aldol reaction approach has been developed and described herein. The key Michael reaction can be accomplished using copper (II)-catalysis of a ketoester and enone to provide an aldol substrate needed for a key carbocyclization to yield a desired complex intermediate. The first chapter will introduce the broad relevance of natural products to medicine and society. Then, tricyclic diterpene natural products that have been described and feature the fused bicyclic scaffold related to isopimaranes will be highlighted. Numerous syntheses that together encapsulate the breadth of synthetic approaches to access the isopimarane-like bioactive scaffolds have been organized and presented herein. Synthetic approaches may mimic natural biosynthesis or be derived from de novo approaches in synthetic connectivity. In the second chapter, the accepted biosynthetic pathway leading to (±)-aspewentin A is presented beginning from common metabolites. A precedent total synthesis will be introduced before the introduction of the novel, concise synthetic approach developed herein. The failed synthetic strategies that provided unexpected, but often in synthetically interesting, carbocycles have also been presented. In the third chapter, the synthetic developments for the Cu(II)-Michael process implementing gem-dimethyl donors, that rapidly enable the formation of a highly congested intermediate are described. Synthetic access to these hindered intermediates enable the concise total synthesis of (±)-aspewentin A.
Pyridoxal 5′-phosphate (PLP)-dependent enzymes are found ubiquitously in nature and are involved in a variety of biological pathways, from natural product synthesis to amino acid and glucose metabolism. The first structure of a PLP-dependent enzyme was reported over 40 years ago, and since that time, there is a steady wealth of structural and functional information revealed for a wide array of these enzymes. A functional mechanism that is gaining more appreciation due to its relevance in drug design is that of protein allostery, where binding of a protein or ligand at a distal site influences the structure, organization, and function at the active site. Here, we present a review of current structure-based mechanisms of allostery for select members of each PLP-dependent enzyme family. Knowledge of these mechanisms may have a larger potential for identifying key similarities and differences among enzyme families that can eventually be exploited for therapeutic development.
Previously, the association between the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) and Dengue virus (DV) replication was demonstrated in liver cells and was found to be mediated at least by the interaction between DDC and phosphoinositide 3-kinase (PI3K). Here, we show that biogenic amines production and uptake impede DV replication in hepatocytes and monocytes, while the virus reduces catecholamine biosynthesis, metabolism, and transport. To examine how catecholamine biosynthesis/metabolism influences DV, first, we verified the role of DDC by altering DDC expression. DDC silencing enhanced virus replication, but not translation, attenuated the negative effect of DDC substrates on the virus and reduced the infection related cell death. Then, the role of the downstream steps of the catecholamine biosynthesis/metabolism was analyzed by chemical inhibition of the respective enzymes, application of their substrates and/or their products; moreover, reserpine, the inhibitor of the vesicular monoamine transporter 2 (VMAT2), was used to examine the role of uptake/storage of catecholamines on DV. Apart from the role of each enzyme/transporter, these studies revealed that the dopamine uptake, and not the dopamine-signaling, is responsible for the negative effect on DV. Accordingly, all treatments expected to enhance the accumulation of catecholamines in the cell cytosol suppressed DV replication. This was verified by the use of chemical inducers of catecholamine biosynthesis. Last, the cellular redox alterations due to catecholamine oxidation were not related with the inhibition of DV replication. In turn, DV apart from its negative impact on DDC, inhibits tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase, and VMAT2 expression.
The rise of various neurodegenerative disorders are somewhat correlating with the worldwide application of multiple anthropogenic toxicants. Though different possible targets were revealed to date, for example, for organophosphorus compounds (OPs), plenty of questions remain. Several decarboxylases (aromatic amino acid decarboxylase, AADC; histidine decarboxylase, HDC; glutamate decarboxylase, GAD) catalyze the biosynthesis of neurotransmitters and neuromodulators and contain pyridoxal phosphate (PLP) as a cofactor. In the current work, 18 OPs which have different neurotoxicity (chemical warfare agents and pesticides) and can penetrate through the blood–brain barrier, were selected. Then, their possible interaction with these decarboxylases in both apo- and holoforms was revealed using computer modeling methods (molecular docking and dynamics). The main amino acid residues of the enzymes responsible for binding OPs have been identified. Individual substances that are most dangerous from the point of view of a possible negative effect on the activity of several decarboxylases were revealed among studied OPs. Glyphosate should be of special interest, since it is not highly toxic towards serine hydrolases, but may prove to be a strong inhibitor for decarboxylases. Holo-AADC could be the most inhibition-prone enzyme among all those investigated.
Dopamine is a catecholamine and an anti-oxidant which functions in responses to stress and it interacts with plant hormones to mediate plant development. At present, there are few studies on the functions of dopamine in apple. This study developed a method for dopamine determination which was used to analyze dopamine in Malus germplasm, in order to clarify the tissue distribution, developmental changes, diurnal variations, and stress responses in apple trees. First, the proposed method was verified. The linear range of quantification was robust from 0.1 to 20 ng mL⁻¹. The instrumental, inter-day precision, and sample repeatability relative standard deviations were 1.024, 5.607, and 7.237%, respectively. The spiked recovery was greater than 100%, indicating the feasibility of the method and its suitability for the rapid analysis of dopamine in Malus. Next, the dopamine content was measured in 322 Malus tissues. The results showed that the dopamine level in Malus was low and the average dopamine content in leaf was higher than in peel and flesh. The dopamine had a skewed distribution that deviated to the right in cultivars and wild accessions. Finally, the tissue specificity, developmental changes, diurnal changes, and responses to stress were analyzed. In cultivar ‘Pinova’ (Malus domestica), the dopamine concentration was the highest in leaf buds and lowest in flesh. The dopamine contents in leaf and flesh decreased with the growth and development of cultivar ‘Liangxiang’ (Malus domestica). The dopamine content of apple leaves was higher after either drought or salinity stress as compared to the control. In this study, a dopamine detection method for apple was established based on HPLC-MS and shown to be a robust approach. This study provides a framework for future research on elucidating the tissue distribution, developmental changes, diurnal variation, and stress responses of dopamine in apple trees.
Background
Aromatic L-amino acid decarboxylase (AADC) deficiency, caused by a pathogenic variant in the dopa decarboxylase (DDC) gene, is a rare neurometabolic disorder in which catecholamine and serotonin are not synthesized. From a large number of reports, it has been recognized that most affected patients show severe developmental delay in a bedridden state and are unable to speak. On the other hand, patients with a mild phenotype with AADC deficiency have been reported, but they number only a few cases. Therefore, the variation of phenotypes of the disease appears to be broad, and it may be challenging to diagnose an atypical phenotype as AADC deficiency.
Case report
We report novel compound heterozygous variants in DDC (c.202G > A and c.254C > T) in two sisters, whose main complaint was mild developmental delay, by whole-exome sequencing (WES). Additionally, we describe their clinical features and provide an image that shows the variants located at different sites responsible for the catalysis of AADC in a three-dimensional structure. The patients were prescribed a Monoamine oxidase (MAO) inhibitor after diagnosis.
Interpretation
Our cases indicate that a comprehensive genomic approach helps to diagnose AADC deficiency with atypical features, and underscore the significance of understanding the variations of this disorder for diagnosis and appropriate treatment.
3-Hydroxytyramine (dopamine) is a neurotransmitter of dopaminergic neurons; in the central nervous system, it plays an essential role in body motor functions. Dopamine formation is part of catecholamine synthesis in which the amino acid tyrosine is finally converted to epinephrine (adrenaline). Dopamine synthesis occurs mainly in dopaminergic systems of the central nervous system, but dopamine is synthetized in other tissues as well, such as the enteric nervous system where tyrosine hydroxylase-immunoreactivity has been observed. Dopamine synthesis includes two reactions, the hydroxylation of the amino acid tyrosine to L-3,4-dihydroxyphenylalanine (L-dopa) catalyzed by tyrosine hydroxylase and the decarboxylation of L-dopa to dopamine catalyzed by aromatic amino acid decarboxylase.
Enzymatic synthesis of biochemical commodities is of upmost importance as it represents a greener alternative to traditional chemical synthesis and provides easier downstream processing strategies compared to fermentation-based processes. A microfluidic system used to optimize the enzymatic production of both levodopa (L-DOPA) and dopamine in both single-step and multistep-reaction sequences with yield of approximately 30 % for L-DOPA production and 70% for dopamine production is presented. The system for L-DOPA production was then up-scaled (780-fold increase) to a milliliter scale system by maintaining similar mass transport properties resulting in the same yield, space-time yield and biocatalyst yield as its microscale counterpart. The results obtained for yield and biocatalyst yield (351.7 mgL-DOPA. mg-1Tyr. h-1) were similar to what is reported in the literature for similar systems, however the space-time yield (0.806 mgL-DOPA.L-1. h-1) was smaller. This work demonstrates a microfluidic bioreactor that can be used for complex optimization that can be performed rapidly while reducing the consumption of reagents by immobilizing the catalyst on a carrier which can then be used in a packed-bed reactor, thus extending the enzyme life span.
Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children.
Human Dopa decarboxylase (hDDC), a pyridoxal 5'-phosphate (PLP) enzyme, displays maxima at 420 and 335 nm and emits fluorescence at 384 and 504 nm upon excitation at 335 nm and at 504 nm when excited at 420 nm. Absorbance and fluorescence titrations of hDDC-bound coenzyme identify a single pKspec of ~7.2. This pKspec could not represent the ionization of a functional group on the Schiff base but that of an enzymic residue governing the equilibrium between the low- and the high-pH forms of the internal aldimine. During the reaction of hDDC with L-Dopa, monitored by stopped-flow spectrophotometry, a 420 nm band attributed to the 4'-N-protonated external aldimine first appears, and its decrease parallels the emergence of a 390 nm peak, assigned to the 4'-N-unprotonated external aldimine. The pH profile of the spectral change at 390 nm displays a pK of 6.4, a value similar to that (~6.3) observed in both k cat and k cat/K m profiles. This suggests that this pK represents the ESH(+) → ES catalytic step. The assignment of the pKs of 7.9 and 8.3 observed on the basic side of k cat and the PLP binding affinity profiles, respectively, is also analyzed and discussed.
To elucidate the role of the synaptic protein α-synuclein in neurodegenerative disorders, transgenic mice expressing wild-type
human α-synuclein were generated. Neuronal expression of human α-synuclein resulted in progressive accumulation of α-synuclein—and
ubiquitin-immunoreactive inclusions in neurons in the neocortex, hippocampus, and substantia nigra. Ultrastructural analysis
revealed both electron-dense intranuclear deposits and cytoplasmic inclusions. These alterations were associated with loss
of dopaminergic terminals in the basal ganglia and with motor impairments. These results suggest that accumulation of wild-type
α-synuclein may play a causal role in Parkinson's disease and related conditions.
Dopa or aromatic amino acid decarboxylase (DDC, AADC) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the production of the neurotransmitters dopamine and serotonin. Among the so far identified mutations associated with AADC deficiency, an inherited rare neurometabolic disease, the S250F mutation is the most frequent one. Here, for the first time, the molecular basis of the deficit of the S250F variant was investigated both in vitro and in cellular systems. Ser250 is not essential for the catalytic activity of the enzyme. However, its mutation to Phe causes a ∼7-fold reduction of catalytic efficiency and a conformational change in the proximity of the mutated residue that is transmitted to the active site. In cellular extracts of E. coli and mammalian cells, both the specific activity and the protein level of the variant decrease with respect to the wild-type. The results with mammalian cells indicate that the mutation does not affect intracellular mRNA levels, and are consistent with a model where S250F undergoes a degradation process via the proteasome, possibly through an ubiquitination process occurring faster than in the wild-type. Overall, biochemical and cell biology experiments show that loss of function of S250F occurs by two distinct but not exclusive mechanisms affecting activity and folding. Importantly, 4-phenylbutirric acid (4-PBA) or, to a major extent, pyridoxine increase the expression level and, in a dose-dependent manner, the decarboxylase specific activity of mutant-expressing cells. This strongly suggests that 4-PBA and/or pyridoxine administration may be of important value in therapy of patients bearing the S250F mutation.
Dopa decarboxylase (DDC) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that by catalyzing the decarboxylation of L-Dopa and L-5-hydroxytryptophan produces the neurotransmitters dopamine and serotonin. The functional properties of pig kidney and human DDC enzymes have been extensively characterized, and the crystal structure of the enzyme in the holo- and apo-forms has been elucidated. DDC is a clinically relevant enzyme since it is involved in Parkinson's disease (PD) and in aromatic amino acid decarboxylase (AADC) deficiency. PD, a chronic progressive neurological disorder characterized by tremor, bradykinesia, rigidity and postural instability, results from the degeneration of dopamine-producing cells in the substantia nigra of the brain. On the other hand, AADC deficiency is a rare debilitating recessive genetic disorder due to mutations in AADC gene leading to the inability to synthesize dopamine and serotonin. Development delay, abnormal movements, oculogyric crises and vegetative symptoms characterize this severe neurometabolic disease. This article is an up to date review of the therapies currently used in the treatment of PD and AADC deficiency as well as of the recent findings that, on one hand provide precious guidelines for the drug development process necessary to PD therapy, and, on the other, suggest an aimed therapeutic approach based on the elucidation of the molecular defects of each variant associated with AADC deficiency.
3,4-Dihydroxyphenylalanine (Dopa) decarboxylase is a stereospecific pyridoxal 5′-phosphate (PLP)-dependent α-decarboxylase
that converts l-aromatic amino acids into their corresponding amines. We now report that reaction of the enzyme with d-5-hydroxytryptophan or d-Dopa results in a time-dependent inactivation and conversion of the PLP coenzyme to pyridoxamine 5′-phosphate and PLP-d-amino acid Pictet-Spengler adducts, which have been identified by high performance liquid chromatography. We also show that
the reaction specificity of Dopa decarboxylase toward aromatic amines depends on the experimental conditions. Whereas oxidative
deamination occurs under aerobic conditions (Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C.
(1996) J. Biol. Chem. 271, 23954–23959; Bertoldi, M., Dominici, P., Moore, P. S., Maras, B., and Borri Voltattorni, C. (1998)Biochemistry 37, 6552–6561), half-transamination and Pictet-Spengler reactions take place under anaerobic conditions. Moreover, we examined
the reaction specificity of nicked Dopa decarboxylase, obtained by selective tryptic cleavage of the native enzyme between
Lys334 and His335. Although this enzymatic species does not exhibit either decarboxylase or oxidative deamination activities, it retains a
large percentage of the native transaminase activity toward d-aromatic amino acids and displays a slow transaminase activity toward aromatic amines. These transamination reactions occur
concomitantly with the formation of cyclic coenzyme-substrate adducts. Together with additional data, we thus suggest that
native Dopa decarboxylase can exist as an equilibrium among “open,” “half-open,” and “closed” forms.
Histamine is an important chemical mediator for a wide variety of physiological reactions. l-Histidine decarboxylase (HDC) is the primary enzyme responsible for histamine synthesis and produces histamine from histidine
in a one-step reaction. In this study, we determined the crystal structure of human HDC (hHDC) complexed with the inhibitor
histidine methyl ester. This structure shows the detailed features of the pyridoxal-5′-phosphate inhibitor adduct (external
aldimine) at the active site of HDC. Moreover, a comparison of the structures of hHDC and aromatic l-amino acid (l-DOPA) decarboxylase showed that Ser-354 was a key residue for substrate specificity. The S354G mutation at the active site
enlarged the size of the hHDC substrate-binding pocket and resulted in a decreased affinity for histidine, but an acquired
ability to bind and act on l-DOPA as a substrate. These data provide insight into the molecular basis of substrate recognition among the group II pyridoxal-5′-phosphate-dependent
decarboxylases.
Dopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i) values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i) values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i) value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.
DOPA decarboxylase, the dimeric enzyme responsible for the synthesis of neurotransmitters dopamine and serotonin, is involved in severe neurological diseases such as Parkinson disease, schizophrenia, and depression. Binding of the pyridoxal-5'-phosphate (PLP) cofactor to the apoenzyme is thought to represent a central mechanism for the regulation of its activity. We solved the structure of the human apoenzyme and found it exists in an unexpected open conformation: compared to the pig kidney holoenzyme, the dimer subunits move 20 Å apart and the two active sites become solvent exposed. Moreover, by tuning the PLP concentration in the crystals, we obtained two more structures with different conformations of the active site. Analysis of three-dimensional data coupled to a kinetic study allows to identify the structural determinants of the open/close conformational change occurring upon PLP binding and thereby propose a model for the preferential degradation of the apoenzymes of Group II decarboxylases.
Plant volatiles (PVs) are lipophilic molecules with high vapor pressure that serve various ecological roles. The synthesis
of PVs involves the removal of hydrophilic moieties and oxidation/hydroxylation, reduction, methylation, and acylation reactions.
Some PV biosynthetic enzymes produce multiple products from a single substrate or act on multiple substrates. Genes for PV
biosynthesis evolve by duplication of genes that direct other aspects of plant metabolism; these duplicated genes then diverge
from each other over time. Changes in the preferred substrate or resultant product of PV enzymes may occur through minimal
changes of critical residues. Convergent evolution is often responsible for the ability of distally related species to synthesize
the same volatile.
One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects.
The hypofrontality theory of the pathogenesis of schizophrenia predicts that cortical lesions cause psychosis. During a search for abnormalities of catecholaminergic neurotransmission in patients with complex partial seizures of the mesial temporal lobe, we discovered an increase of the rate of metabolism of an exogenous dopa tracer (6-[18F]fluoro-L-dopa) in the neostriatum of a subgroup of patients with a history of psychosis. When specifically assayed for this abnormality, patients with schizophrenia revealed the same significant increase of the rate of metabolism in the striatum. The finding is consistent with the theory that a state of psychosis arises when episodic dopamine excess is superimposed on a trait of basic dopamine deficiency in the striatum. The finding is explained by the hypothesis that cortical insufficiency, a proposed pathogenetic mechanism of both disorders, causes an up-regulation of the enzymes responsible for dopa turnover in the neostriatum as well as the receptors mediating dopaminergic neurotransmission.
L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To advance structure-function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the later protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the Vmax was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme-apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour.
1. Rhythmic activity was induced with either serotonin (5-HT; 10-100 microM) or dopamine (0.1-1.0 mM), in the in vitro spinal cord preparation of neonatal rats, with one intact hindlimb attached. Patterns of activity were investigated with multiple EMG recordings and the spatiotemporal characteristics of 5-HT- and dopamine-induced activity compared. 2. Dopamine-induced rhythmic activity was slow (cycle duration: 2.2-70.1 s) and irregular, whereas rhythmic activity induced by 5-HT was fast (cycle duration: 1.3-5.1 s) and regular. 3. During 5-HT- and dopamine-induced rhythmic activity, the timing of muscular activity was similar for hip flexors and hip adductors, for semimembranosus (hip extensor), and for muscles controlling the ankle and the foot. 4. In contrast, notable differences in the phase in the pattern induced by 5-HT compared with that induced by dopamine were found in the biceps femoris, semitendinosus, and quadriceps muscles. Biceps femoris and semitendinosus (functional hip extensors and knee flexors) were always extensor-like during 5-HT-induced activity, whereas in dopamine, these muscles displayed flexor-like bursts and double bursts as well as extensor-like bursts. Lack of EMG activity in biceps femoris and semitendinosus was encountered also in dopamine. In rectus femoris, vastus lateralis, and vastus medialis (main function: knee extension), the activity was dominated by flexor-like bursts in 5-HT, whereas in dopamine the activity was shifted to a predominantly extensor-like pattern. 5. The relationship between flexor and extensor burst duration and cycle duration was more variable than described for locomotor activity in adult animals. 6. The relative timing of muscle activity was stable from P0 to P4. The most important difference between rats aged 0-1 days and rats aged 2-4 days was a delayed flexor-extensor transition in older animals. 7. The complex timing of hindlimb muscle activity was relatively unchanged after transecting all dorsal roots. 8. Finally, the relationship between flexor and extensor activity and ventral root discharges was determined. It was found that the L2 ventral root burst was in phase with simple flexors while the L5 burst coincide with the extensor phase. 9. We conclude, that 5-HT and dopamine can activate spinal central pattern generators (CPGs) that already at birth are able to produce distinct patterns of motor activity. Modulatory inputs thus seems to be able to reconfigure the CPGs to produce specific motor outputs.
Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5′-phosphate active site per subunit. In addition to
catalyzing the decarboxylation of L-aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammonia.
These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with
high performance liquid chromatographic and mass spectroscopic analysis. The Kcat and Km values of this reaction were determined to be 0.48 min−1 and 0.47 mM, respectively. The NaBH4-reduced enzyme does not catalyze this reaction. Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent
manner and exhibits saturation of the rate of inactivation at high concentrations, with Ki and Kinact values of 0.40 mM and 0.023 min−1, respectively. Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine. Inactivation with [2-14C]5-HT results in the incorporation of 1 mol of label/enzyme subunit. Taken together, these findings indicate that 5-HT is
both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21. The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized. A
speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented.
DOPA decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase (AADC), is an enzyme involved in the synthesis of the important neurotransmitters dopamine, norepinephrine, and serotonin. In addition, it participates in the synthesis of trace amines; compounds suggested to act as endogenous modulators of central neurotransmission. Thus, DDC is regarded as a potential susceptibility gene for a variety of neuropsychiatric disorders. The aim of the present study was to examine the role of DDC in bipolar affective disorder (BPAD). By screening 10 individuals for sequence variations in the coding region of the DDC gene as well as in the neuron-specific promoter and 5' untranslated regions we were able to identify two fairly frequent variants: a 1-bp deletion in the promoter and a 4-bp deletion in the untranslated exon 1. Both deletions affect putative binding sites for known transcription factors, suggesting a possible functional impact at the level of expression. The two variants were applied in an association study including 80 Danish bipolar patients, 112 English bipolar patients, 223 Danish controls, and 349 English controls. Analyzing the combined material, a significant association was found between the 1-bp deletion and BPAD with P-values of 0.037 (allelic) and 0.021 (genotypic). The frequency of the 1-bp deletion was 13.3% in patients and 9.4% in controls with a corresponding odds ratio of 1. 48 (95% CI: 1.02-2.15). The results presented suggest that DDC may act as a minor susceptibility gene for bipolar affective disorder.
DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinson's disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.
A flexible loop (residues 328-339), presumably covering the active site upon substrate binding, has been revealed in 3,4-dihydroxyphenylalanine decarboxylase by means of kinetic and structural studies. The function of tyrosine 332 has been investigated by substituting it with phenylalanine. Y332F displays coenzyme content and spectroscopic features identical to those of the wild type. Unlike wild type, during reactions with l-aromatic amino acids under both aerobic and anaerobic conditions, Y332F does not catalyze the formation of aromatic amines. However, analysis of the products shows that in aerobiosis, l-aromatic amino acids are converted into the corresponding aromatic aldehydes, ammonia, and CO(2) with concomitant O(2) consumption. Therefore, substitution of Tyr-332 with phenylalanine results in the suppression of the original activity and in the generation of a decarboxylation-dependent oxidative deaminase activity. In anaerobiosis, Y332F catalyzes exclusively a decarboxylation-dependent transamination of l-aromatic amino acids. A role of Tyr-332 in the Calpha protonation step that catalyzes the formation of physiological products has been proposed. Furthermore, Y332F catalyzes oxidative deamination of aromatic amines and half-transamination of d-aromatic amino acids with k(cat) values comparable with those of the wild type. However, for all the mutant-catalyzed reactions, an increase in K(m) values is observed, suggesting that Y --> F replacement also affects substrate binding.
Glutamate decarboxylase is a vitamin B6-dependent enzyme, which catalyses the decarboxylation of glutamate to gamma-aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH-dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure-based site-directed mutagenesis that the triple helix bundle formed by the N-termini of the protein at acidic pH is the major determinant for this behaviour.
It is widely appreciated that the selection and modulation of locomotor circuits are dependent on the actions of higher-order projection neurons. In the leech, Hirudo medicinalis, locomotion is modulated by a number of cephalic projection neurons that descend from the subesophageal ganglion in the head. Specifically, descending brain interneuron Tr2 functions as a command-like neuron that can terminate or sometimes trigger fictive swimming. In this study, we demonstrate that Tr2 is dye coupled to the dopaminergic neural network distributed in the head brain. These findings represent the first anatomical evidence in support of dopamine (DA) playing a role in the modulation of locomotion in the leech. In addition, we have determined that bath application of DA to the brain and entire nerve cord reliably and rapidly terminates swimming in all preparations exhibiting fictive swimming. By contrast, DA application to nerve cords expressing ongoing fictive crawling does not inhibit this motor rhythm. Furthermore, we show that Tr2 receives rhythmic feedback from the crawl central pattern generator. For example, Tr2 receives inhibitory post-synaptic potentials during the elongation phase of each crawl cycle. When crawling is not expressed, spontaneous inhibitory post-synaptic potentials in Tr2 correlate in time with spontaneous excitatory post-synaptic potentials in the CV motor neuron, a circular muscle excitor that bursts during the elongation phase of crawling. Our data are consistent with the idea that DA biases the nervous system to produce locomotion in the form of crawling.
The green tea gallocatechins, (−)-epigallocatechin-3-O-gallate (EGCG), and (−)-epigallocatechin (EGC) were found to be inhibitors of Dopa decarboxylase (DDC). EGCG and EGC inactivate the enzyme in both a time- and concentration-dependent manner and exhibit saturation of the rate of inactivation at high concentrations, with efficiency of inactivation values (kinact/Ki) of 868 and 1511 M−1 min−1, respectively. In contrast, gallic acid behaves as a weak inhibitor of DDC. Protection against inactivation by EGCG and EGC was observed in the presence of the active site-directed inhibitor D-Dopa. Either EGCG or EGC induce changes in the absorbance and CD bands of the visible spectrum of enzyme-bound PLP. Taken together, these findings indicate the active site nature of the interaction of DDC with both polyphenols. On the basis of the properties of the EGCG-inactivated enzyme, it can be suggested that inactivation could be ascribed to a covalent modification of not yet identified residue(s) of the active site of DDC.
Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T(3). TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the L-alanine side chain and several deiodination steps to convert e.g. L-thyroxine (T(4)) into the most potent 3-T(1)AM. Aromatic L-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T(1)AM in plasma of patients with AADC-deficiency at levels (46 ± 18 nM, n=4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase.
Dopa decarboxylase (DDC) catalyzes the cleavage of alpha-methylDopa into 3,4-dihydroxyphenylacetone and ammonia, via the intermediate alpha-methyldopamine, which does not accumulate during catalysis. The ketone has been identified by high-performance liquid chromatography and mass spectroscopic analysis, and ammonia by means of glutamate dehydrogenase. Molecular oxygen is consumed during the reaction in a 1:2 molar ratio with respect to the products. The kcat and Km of this reaction were determined to be 5.68 min-1 and 45 microM, respectively. When the reaction is carried out under anaerobic conditions, alpha-methyldopamine is formed in a time-dependent manner and neither ammonia nor ketone is produced to a significant extent. The reaction is accompanied by a time- and concentration-dependent inactivation of the enzyme with kinact of 0. 012 min-1 and Ki of 39.3 microM. Free 3,4-dihydroxyphenylacetone binds to the active site of DDC and inactivates the enzyme in a time- and concentration-dependent manner with a kinact/Ki value similar to that of alpha-methylDopa. d-Dopa, a competitive inhibitor of DDC, protects the enzyme against inactivation. Taken together, these findings indicate the active site directed nature of the interaction of DDC with 3,4-dihydroxyphenylacetone and provide evidence that the ketone generated by the reaction of DDC with alpha-methylDopa dissociates from the active site before it inactivates the enzyme. Inactivation of the enzyme by ketone followed by NaB3H4 reduction and chymotryptic digestion revealed that the lysine residue which binds pyridoxal 5'-phosphate (PLP) in the native enzyme is the site of covalent modification. Together with the characterization of the adduct released from the inactivated DDC, these data suggest that the enzyme is inactivated by trapping the coenzyme in a ternary adduct with ketone and the active site lysine. As recently reported for serotonin (5-HT) [Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol. Chem. 271, 23954-23959], the conversion of dopamine (DA) into 3,4-dihydroxyphenylacetaldehyde and ammonia catalyzed by DDC is accompanied by irreversible loss of decarboxylase activity. However, the comparison between the absorbance, fluorescence, and CD features of DDC after 5-HT- or 3, 4-dihydroxyphenylacetone-induced inactivation shows that a different covalent adduct is formed between either of these two molecules and DDC-bound PLP.
Dopa decarboxylase (DDC or AADC) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-aromatic amino acids into the corresponding aromatic amines. AADC deficiency is an inborn error of neurotransmitters biosynthesis with an autosomal recessive inheritance. About 30 pathogenic mutations have been identified, but the enzymatic phenotypes causing AADC deficiency are unknown, and the therapeutic management is challenging. Here, we report biochemical and bioinformatic analyses of the human wild-type DDC and the pathogenic variants G102S, F309L, S147R and A275T whose mutations concern amino acid residues at or near the active site. We found that the mutations cause, even if to different extents, a decreased PLP binding affinity (in the range 1.4-170-fold), an altered state of the bound coenzyme and of its microenvironment, and a reduced catalytic efficiency (in the range 17-930-fold). Moreover, as compared to wild-type, the external aldimines formed by the variants with L-aromatic amino acids exhibit different spectroscopic features, do not protect against limited proteolysis, and lead to the formation, in addition to aromatic amines, of cyclic-substrate adducts. This suggests that these external Schiff bases are not properly oriented and anchored, i.e., in a conformation not completely productive for decarboxylation. The external aldimines that the variants form with D-Dopa also appear not to be correctly located at their active site, as suggested by the rate constants of PLP-L-Dopa adduct production higher than that of the wild-type. The possible therapeutic implications of the data are discussed in the light of the molecular defects of the pathogenic variants.
2-Phenylethanol (2PE) is a prominent scent compound released from flowers of Damask roses (Rosa×damascena) and some hybrid roses (Rosa 'Hoh-Jun' and Rosa 'Yves Piaget'). 2PE is biosynthesized from l-phenylalanine (l-Phe) via the intermediate phenylacetaldehyde (PAld) by two key enzymes, aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). Here we describe substrate specificity and cofactor preference in addition to molecular characterization of rose-PAR and recombinant PAR from R.×damascena. The deduced amino acid sequence of the full-length cDNA encoded a protein exhibiting 77% and 75% identity with Solanum lycopersicum PAR1 and 2, respectively. The transcripts of PAR were higher in petals than calyxes and leaves and peaking at the unfurling stage 4. Recombinant PAR and rose-PAR catalyzed reduction of PAld to 2PE using NADPH as the preferred cofactor. Reductase activity of rose-PAR and recombinant PAR were higher for aromatic and aliphatic aldehydes than for keto-carbonyl groups. Both PARs showed that (S)-[4-(2)H] NADPH was preferentially used over the (R)-[4-(2)H] isomer to give [1-(2)H]-2PE from PAld, indicating that PAR can be classified as short-chain dehydrogenase reductase (SDR).
The pyridoxal 5'-phosphate dependent-enzyme Dopa decarboxylase, responsible for the irreversible conversion of l-Dopa to dopamine, is an attractive drug target. The contribution of the pyridoxal-Lys303 to the catalytic mechanisms of decarboxylation and oxidative deamination is analyzed. The K303A variant binds the coenzyme with a 100-fold decreased apparent equilibrium binding affinity with respect to the wild-type enzyme. Unlike the wild-type, K303A in the presence of l-Dopa displays a parallel progress course of formation of both dopamine and 3,4-dihydroxyphenylacetaldehyde (plus ammonia) with a burst followed by a linear phase. Moreover, the finding that the catalytic efficiencies of decarboxylation and of oxidative deamination display a decrease of 1500- and 17-fold, respectively, with respect to the wild-type, is indicative of a different impact of Lys303 mutation on these reactions. Kinetic analyses reveal that Lys303 is involved in external aldimine formation and hydrolysis as well as in product release which affects the rate-determining step of decarboxylation.
Levodopa has been the mainstay of treatment for Parkinson's disease (PD) for more than 40 years. During this time, researchers have strived to optimize levodopa formulations to minimize side effects, enhance central nervous system (CNS) bioavailability, and achieve stable therapeutic plasma levels. Current strategies include concomitant treatment with inhibitors of dopa decarboxylase (DDC) and catechol-O-methyltransferase (COMT) to prolong the peripheral levodopa half-life and increase CNS bioavailability. Levodopa combined with DDC inhibition is the current standard method of delivering levodopa for symptomatic treatment of PD. Recent research suggests that continuous dopaminergic stimulation that more closely approximates physiological stimulation may delay or prevent the development of motor fluctuations ('wearing off') and dyskinesias. Strategies currently being used to achieve more continuous dopaminergic stimulation include the combination of an oral levodopa/DDC inhibitor with a COMT inhibitor and the enteral infusion of a levodopa gel formulation. Attempts are underway to develop oral and transdermal very long-acting levodopa preparations.
A simple and rapid procedure, which takes advantage of the effectiveness of conventional and HPLC hydrophobic interaction, for the isolation of highly purified rat liver 3,4-dihydroxyphenylalanine decarboxylase is described in detail.
Some of its structural and functional properties are reported and discussed in comparison with those of pig kidney 3,4-dihydroxyphenylalanine decarboxylase.
Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase can be nicked by trypsin with complete loss of its catalytic activity. The original dimer of subunit molecular weight of about 52,000 yields fragments of Mr 38,000 and 14,000, as seen on sodium dodecyl sulfate-gel electrophoresis. Though inactive, the nicked protein retains its native molecular weight and its capacity to bind pyridoxal-5'-phosphate (pyridoxal-P), is recognized by an antiserum raised against the native enzyme, and forms Schiff's base intermediates with aromatic amino acids in L and D forms. Thus, the nicked protein appears to be in a conformation--closely resembling that of the original enzyme--which consists of a tight association of the two tryptic fragments. Dissociation and separation of the two fragments can be achieved under denaturing conditions on a reverse-phase HPLC column. The pyridoxal-P binding site is located on the larger fragment. No NH2-terminal residue is detected in either the intact enzyme or the larger fragment, whereas analysis of the smaller fragment yields a sequence of the first 50 amino acid residues. These data indicate that the smaller fragment is located at about one-third from the COOH terminus of Dopa decarboxylase, while the larger fragment constitutes the aminic portion of the molecule. The site of trypsin cleavage seems to be in a region of the enzyme particularly susceptible to proteolysis. The results of these studies contribute to a better understanding of the structural properties of pig kidney Dopa decarboxylase and may constitute an important step toward the elucidation of the enzyme's primary structure.
L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.
Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bound at the active site with great affinity. From a comparison of the interaction of a number of such compounds with the apoenzyme the delta G0 values for the binding of the substrate carboxy and phenyl groups and of the coenzyme aldehydic group were determined to be equal to (or more negative than) -3.8. -8.4 and -12.5kJ/mol (-0.9, -1.9 and -3kcal/mol) respectively; the delta G0 for the binding of the coenzyme phosphate group was shown to be more negative than -20.5kJ/mol (-4.9kcal/mol). Two features of the binding process of the coenzyme-substrate analogues to tyrosine decarboxylase have already been found in the case of tyrosine aminotransferase [Borri-Voltattorni, Orlacchio, Giartosio, Conti & Turano (1975) Eur. J. Biochem. 53, 151-160]: (1) in the binding of the substrate to the enzyme a significant fraction of the instrinsic delta G0 appears to be used for some associated endoergonic process; (2) the delta H0 and delta S0 of binding appear to be very sensitive indicators of the correct alignment of the substrate-coenzyme and analogues at the active site.
The two pyloric dilator (PD) motor neurons and the single anterior burster (AB) interneuron are electrically coupled and together comprise the pacemaker for the pyloric central pattern generator of the stomatogastric ganglion of the lobster, Panulirus interruptus. Previous work (31) has shown that the AB neuron is an endogenously bursting neuron, while the PD neuron is a conditional burster. In this paper the effects of physiological inputs and neurotransmitters on isolated PD neurons and AB neurons were studied using the lucifer yellow photoinactivation technique (33). Stimulation of the inferior ventricular nerve (IVN) fibers at high frequencies elicits a triphasic response in AB and PD neurons: a rapid excitatory postsynaptic potential (EPSP) followed by a slow inhibitory postsynaptic potential (IPSP), followed by an enhancement of the pacemaker slow-wave depolarizations. Photoinactivation experiments indicate that the enhancement of the slow wave is due primarily to actions of the IVN fibers on the PD neurons but not on the AB neuron. Bath-applied dopamine dramatically alters the motor output of the pyloric system. Photoinactivation experiments show that 10(-4) M dopamine increases the amplitude and frequency of the slow-wave depolarizations recorded in the AB neurons but hyperpolarizes and inhibits the PD neurons. Bath-applied serotonin increases the frequency and amplitude of the slow-wave depolarizations in the AB neuron but has no effect on PD neurons. Pilocarpine, a muscarinic cholinergic agonist, stimulates slow-wave depolarization production in both PD neurons and the AB neuron, but the waveform and frequency of the slow waves elicited are quite different. These results show that although the electrically coupled PD and AB neurons always depolarize synchronously and act together as the pacemaker for the pyloric system, they respond differently to a neuronal input and to several putative neuromodulators. Thus, despite electrical coupling sufficient to ensure synchronous activity, the PD and AB neurons can be modulated independently.
The actions exerted by aromatic D-amino acids on 3,4-dihydroxyphenylalanine (Dopa) decarboxylase have been examined by measuring their effects on the enzymic activity and on the spectral properties of the pyridoxal 57prime;-phosphate bound enzyme. It has been shown that Dopa decarboxylase does not decarboxylate aromatic D-amino acids, thus indicating its stereospecificity. However, the enzyme does appear to bind these amino acids at the active site. Dopa, m-tyrosine, and o-tyrosine in D forms appear to inhibit the enzymic activity and to form stable intermediate complexes absorbing at 420 nm with the enzyme. 5-Hydroxytryptophan and tryptophan in D forms appear to exert a time-dependent inactivation and to form with the enzyme intermediate complexes absorbing at 420 nm undergoing time-dependent modification; the formation of pyridoxamine 5′-phosphate is also observed. The behavior of these latter two amino acids can be partially attributed to a transamination catalyzed by Dopa decarboxylase; a plausible mechanism of inactivation has not yet been elucidated. The interaction of the aromatic amino acids in D forms with the enzymes calls for critical revision of all previously obtained results by using the racemic forms as substrates. Therefore we here report on the kinetic parameters for Dopa, 5-hydroxytryptophan, w-tyrosine, and o-tyrosine in L forms, showing that unnatural substrates have a decarboxylation rate higher than that of the natural ones. We have also identified two enzyme-substrate intermediate complexes absorbing at 420 and 390 nm which are displayed by the low and high pH forms of the complexes, respectively. Considerations on the nature of this spectral change have been reported. The different pK value of the shift 420-390 nm, as determined for L-Dopa and L-5-hydroxytryptophan, appears to be related to the different chemical structure of the substrate. The authors also discuss the relevance of their findings toward a better elucidation of Dopa decarboxylase active site.
Two of the five domains in the structure of the ornithine decarboxylase (OrnDC) from Lactobacillus 30a share similar structural folds around the pyridoxal-5'-phosphate (PLP)-binding pocket with the aspartate aminotransferases (AspATs). Sequence comparisons focusing on conserved residues of the aligned structures reveal that this structural motif is also present in a number of other PLP-dependent enzymes including the histidine, dopa, tryptophan, glutamate, and glycine decarboxylases as well as tryptophanase and serine-hydroxymethyl transferase. However, this motif is not present in eukaryotic OrnDCs, the diaminopimelate decarboxylases, nor the Escherichia coli or oat arginine decarboxylases. The identification and comparison of residues involved in defining the different classes are discussed.
Neurons in the central nervous system (CNS) often store more than one neurotransmitter, but as yet the functional significance of this type of coexistence is poorly understood. 5-Hydroxytryptamine (5-HT) modulates calcium-dependent K+ channels (KCa) responsible for the postspike afterhyperpolarization in different regions of the CNS. In lamprey, 5-HT neurons control apamine-sensitive KCa channels in spinal locomotor network interneurons, thereby in addition regulating the duration of locomotor bursts. We report here that these spinal 5-HT neurons also contain dopamine. Like 5-HT, dopamine causes a reduction of the afterhyperpolarization, but in this case it is due to a reduction of calcium entry during the action potential, which results in a reduced activation of KCa. 5-HT and dopamine are both released from these midline neurons, and both reduce the afterhyperpolarization through two distinctly different, but complementary cellular mechanisms. The net effect of dopamine (10-100 microM) on the locomotor network is similar to that of 5-HT, and the effects of dopamine and 5-HT are additive at the network level.
Pharmacotherapy with levodopa for Parkinson's disease provides symptomatic benefit, but fluctuations in (or loss of) response may eventually occur. Dopamine agonists are also helpful and, when taken with low doses of levodopa, often provide sustained benefit with fewer side effects; novel agonists and new methods for their administration are therefore under study. Other therapeutic strategies are being explored, including the use of type B monoamine oxidase inhibitors to reduce the metabolic breakdown of dopamine, catechol-O-methyltransferase inhibitors to retard the breakdown of levodopa, norepinephrine precursors to compensate for deficiency of this neurotransmitter, glutamate antagonists to counteract the effects of the subthalamic nucleus, and various neurotrophic factors to influence dopaminergic nigrostriatal cells. Surgical procedures involving pallidotomy are sometimes helpful. Those involving cerebral transplantation of adrenal medullary or fetal mesencephalic tissue have yielded mixed results; benefits may relate to the presence of growth factors in the transplanted tissue. The transplantation of genetically engineered cell lines will probably become the optimal transplantation procedure. The cause of Parkinson's disease may relate to oxidant stress and the generation of free radicals. It is not clear whether treatment with selegiline hydrochloride (a type B monoamine oxidase inhibitor) delays the progression of Parkinson's disease, because the drug also exerts a mild symptomatic effect. Daily treatment with vitamin E (a scavenger of free radicals) does not influence disease progression, perhaps because of limited penetration into the brain.
The physiochemical properties of the coenzyme in rat liver aromatic L-amino acid decarboxylase (AADC) expressed in Escherichia coli have been studied by spectroscopic analysis of the enzyme, its reaction intermediates, and its complexes with substrate analogs. The enzyme, having one pyridoxal 5'-phosphate (PLP) per subunit, shows a prominent absorption maximum at 335 nm and a weaker one at 425 nm. The spectrum did not essentially change in the pH range of 6.0-8.0. When the coenzyme was excited at 335 nm, it emitted fluorescence primarily at 520 nm. The structure for the absorption at 335 nm was ascribed to the enolimine form of the PLP-lysine Schiff base. On the reaction of AADC with L-3,4-dihydroxyphenylalanine (L-dopa), the absorption of PLP showed biphasic changes before reaching a steady-state. Results of both pre-steady-state and steady-state kinetic analyses were consistent with the model that the reaction proceeds as shown in the equation: E + S<==>X1<==>X2-->E + P. The rate constant was determined for each step, and the Km value for L-dopa was obtained as 0.086 mM. The absorption spectra of the two intermediates, X1 and X2, were postulated from the calculation of the absorption changes during the first and the second steps of the reaction in which X1 and X2 showed an absorption maximum at 425 and 380 nm, respectively, with a concomitant decrease in absorbance at 335 nm. These predicted absorption spectra of X1 and X2 showed striking resemblances to those of AADC complexed with dihydroxyphenylacetic acid (DOPAc) and L-dopa methyl ester (DopaOMe), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Transient and steady-state kinetic analysis of the reaction of aromatic L-amino acid decarboxylase (AADC), a pyridoxal 5'-phosphate- (PLP-) dependent enzyme, with its substrate dopa was carried out at various pH. The association of AADC and dopa to form the Michaelis complex and the subsequent transaldimination reaction to form the dopa-PLP Schiff base (external aldimine) were followed with a stopped-flow spectrophotometer. Combined with the steady-state k(cat) value, we could present a minimum mechanism for the reaction of AADC and dopa. In the mechanism, the association of the aldimine-protonated form of the enzyme (EH(+)) and the alpha-amino-group-unprotonated form of the substrate (S) is the main route leading to the Michaelis complex. In addition, the association of EH(+) and the alpha-amino-group-protonated form of the substrate (SH(+)) to form a Michaelis complex EH(+).SH(+) was also found as a minor route. The pK(a) of the alpha-amino group of dopa was expected to be decreased in the Michaelis complex, promoting the conversion of EH(+).SH(+) to EH(+).S, the species that directly undergoes transaldimination to form the external aldimine complex. The association of EH(+) and S had been identified as a minor route for the reaction of aspartate and aspartate aminotransferase (AspAT), which has an unusually low pK(a) value of the aldimine and can use the aldimine-unprotonated form (E) of the enzyme for adsorbing the prevalent species SH(+) [Hayashi and Kagamiyama (1997) Biochemistry 36, 13558-13569]. The present study implies that, in most PLP enzymes that have a high pK(a) value of the aldimine like AADC, S preferentially binds to the enzyme (EH(+)). The minor route of EH(+) + SH(+) in AADC may be related to the flexibility of the protein in the Michaelis complex, and a simulation analysis showed that the presence of this route decreases the k(cat) value while increasing the k(cat)/K(m) value. It also suggested that AADC has evolved to suppress the minor route to the extent necessary to obtain the maximal k(cat) value at neutral pH.
Parkinson's disease is a common neurodegenerative syndrome characterized
by loss of dopaminergic neurons in the substantia nigra, formation of filamentous
intraneuronal inclusions (Lewy bodies) and an extrapyramidal movement disorder.
Mutations in the -synuclein gene are linked to familial Parkinson's
disease1, 2 and -synuclein accumulates in Lewy bodies
and Lewy neurites3, 4, 5. Here we express normal and mutant
forms of -synuclein in Drosophila and produce adult-onset loss
of dopaminergic neurons, filamentous intraneuronal inclusions containing -synuclein
and locomotor dysfunction. Our Drosophila model thus recapitulates
the essential features of the human disorder, and makes possible a powerful
genetic approach to Parkinson's disease.
A comprehensive survey of the extensive literature relevant to the evolution, physiology, biochemistry, regulation, and genetic engineering applications of plant aromatic L-amino acid decarboxylases (AADCs) is presented. AADCs catalyze the pyridoxal-5'-phosphate (PLP)-dependent decarboxylation of select aromatic L-amino acids in plants, mammals, and insects. Two plant AADCs, L-tryptophan decarboxylase (TDC) and L-tyrosine decarboxylase (TYDC), have attracted considerable attention because of their role in the biosynthesis of pharmaceutically important monoterpenoid indole alkaloids and benzylisoquinoline alkaloids, respectively. Although plant and animal AADCs share extensive amino acid homology, the enzymes display striking differences in their substrate specificities. AADCs from mammals and insects accept a broad range of aromatic L-amino acids, whereas TDC and TYDC from plants exhibit exclusive substrate specificity for L-amino acids with either indole or phenol side chains, but not both. Recent biochemical and kinetic studies on animal AADCs support basic features of the classic AADC reaction mechanism. The catalytic mechanism involves the formation of a Schiff base between PLP and an invariable lysine residue, followed by a transaldimination reaction with an aromatic L-amino acid substrate. Both TDC and TYDC are primarily regulated at the transcriptional level by developmental and environmental factors. However, the putative post-translational regulation of TDC via the ubiquitin pathway, by an ATP-dependent proteolytic process, has also been suggested. Isolated TDC and TYDC genes have been used to genetically alter the regulation of secondary metabolic pathways derived from aromatic amino acids in several plant species. The metabolic modifications include increased serotonin levels, reduced indole glucosinolate levels, redirected shikimate metabolism, increased indole alkaloid levels, and increased cell wall-bound tyramine levels.
Analysis of the reaction of dopa decarboxylase (DDC) with L-dopa reveals that loss of decarboxylase activity with time is observed at enzyme concentrations approximately equal to the binding constant, K(d), of the enzyme for pyridoxal 5'-phosphate (PLP). Instead, at enzyme concentrations higher than K(d) the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5'-phosphate (PMP) is formed during the reaction and that dissociation of coenzyme occurs at low enzyme concentration, leading to the formation of a PLP-L-dopa Pictet-Spengler cyclic adduct. Taken together, these results indicate that decarboxylation-dependent transamination does not accompany the decarboxylation of L-dopa proposed previously [O'Leary and Baughn (1977) J. Biol. Chem. 252, 7168-7173]. Nevertheless, when the reaction of DDC with L-dopa is studied under anaerobic conditions at an enzyme concentration higher than K(d), we observe that (1) the enzyme is gradually inactivated and inactivation is associated with PMP formation and (2) the initial velocity of decarboxylation is approximately half of that in the presence of O(2). Similar behaviour is observed by comparing the reaction with L-5-hydroxytryptophan occurring in aerobiosis or in anaerobiosis. Therefore the reaction of DDC with L-aromatic amino acids seems to be under O(2) control. In contrast, the reactivity of the enzyme with L-aromatic amino acids does not change in the presence or absence of O(2). These and other results, together with previous results on the effect exerted by O(2) on reaction specificity of DDC towards aromatic amines [Bertoldi, Frigeri, Paci and Borri Voltattorni (1999) J. Biol. Chem. 274, 5514-5521], suggest a productive effect of O(2) on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines.
Residues D271, H192, H302 and N300 of L-3,4-dihydroxyphenylalanine decarboxylase (DDC), a homodimeric pyridoxal 5'-phosphate (PLP) enzyme, were mutated in order to acquire information on the catalytic mechanism. These residues are potential participants in catalysis because they belong to the common PLP-binding structural motif of group I, II and III decarboxylases and other PLP enzymes, and because they are among the putative active-site residues of structural modelled rat liver DDC. The spectroscopic features of the D271E, H192Q, H302Q and N300A mutants as well as their dissociation constants for PLP suggest that substitution of each of these residues causes alteration of the state of the bound coenzyme molecule and of the conformation of aromatic amino acids, possibly in the vicinity of the active site. This supports, but does not prove, the possibility that these residues are located in the coenzyme-binding cleft. Interestingly, mutation of each residue generates an oxidative decarboxylase activity towards L-3,4-dihydroxyphenylalanine (L-Dopa), not inherent in the wild-type in aerobiosis, and reduces the nonoxidative decarboxylase activity of L-Dopa from 3- to 390-fold. The partition ratio between oxidative and nonoxidative decarboxylation ranges from 5.7 x 10(-4) for N300A mutant to 946 x 10(-4) for H302Q mutant. Unlike wild-type enzyme, the mutants catalyse these two reactions to the same extent either in the presence or absence of O2. In addition, all four mutants exhibit an extremely low level of the oxidative deaminase activity towards serotonin with respect to wild-type. All these findings demonstrate that although D271, H192, H302 and N300 are not essential for catalysis, mutation of these residues alters the nature of catalysis. A possible relationship among the integrity of the PLP cleft, the productive binding of O2 and the transition to a closed conformational state of DDC is discussed.
Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.
Dopa decarboxylase (DDC) catalyzes as main reaction the stereospecific CO(2) abstraction from L-Dopa and L-5-hydroxytryptophan (5-HTP), generating the corresponding aromatic amines, dopamine and serotonin, respectively. Side reactions with turnover time of minutes are also catalyzed by the enzyme. In particular, DDC exhibits half-transaminase activity toward D-aromatic amino acids and oxidative deaminase activity toward aromatic amines. The latter reaction could represent a new activity for this class of enzymes. Studies on the effect exerted by O(2) on reaction specificity of DDC revealed that under anaerobic conditions decarboxylation of L-aromatic amino acids takes place with a k(cat) approximately half of that measured in the presence of O(2), and is accompanied by a decarboxylation-dependent transamination, whereas oxidative deamination of aromatic amines is replaced by half-transamination. Half-transamination of D-aromatic amino acids is unaffected by the presence or absence of O(2). Some structural elements relevant for the control of reaction and substrate specificity of DDC have been identified by means of limited tryptic digestion and site-directed mutagenesis studies. All together, the data indicate that the chemical nature of the substrate, the presence of O(2), the integrity of a mobile loop, the absence of perturbation in the coenzyme-binding cleft and pH are important requirements for the achievement of a closed conformational state where the highest level of reaction specificity is reached.
The reactions of Dopa decarboxylase (DDC) with l- and d-enantiomers of tryptophan methyl ester are described. Although both the enantiomers bind to the active site of the enzyme with similar affinity, their binding modes are different. l-enantiomer binds in an unproductive mode, while d-enantiomer acts as an oxidative deamination substrate. For the first time a quinonoid has been detected as intermediate of this reaction. By using rapid-scanning stopped-flow kinetic technique rate constants for formation and decay of this species have been determined. All these data, besides validating the functional DDC active site model, represent an important step toward the elucidation of the catalytic pathway of oxidative deamination.
Tyr 64, hydrogen-bonded to coenzyme phosphate in Treponema denticola cystalysin, was changed to alanine by site-directed mutagenesis. Spectroscopic and kinetic properties of the Tyr 64 mutant were investigated in an effort to explore the differences in coenzyme structure and kinetic mechanism relative to those of the wild-type enzyme. The wild type displays coenzyme absorbance bands at 418 and 320 nm, previously attributed to ketoenamine and substituted aldamine, respectively. The Tyr 64 mutant exhibits absorption maxima at 412 and 325 nm. However, the fluorescence characteristics of the latter band are consistent with its assignment to the enolimine form of the Schiff base. pK(spec) values of approximately 8.3 and approximately 6.5 were observed in a pH titration of the wild-type and mutant coenzyme absorbances, respectively. Thus, Tyr 64 is probably the residue involved in the nucleophilic attack on C4' of pyridoxal 5'-phosphate (PLP) in the internal aldimine. Although the Tyr 64 mutant exhibits a lower affinity for PLP and lower turnover numbers for alpha,beta-elimination and racemization than the wild type, the pH profiles for their Kd(PLP) and kinetic parameters are very similar. Rapid scanning stopped-flow and chemical quench experiments suggest that, in contrast to the wild type, for which the rate-determining step of alpha,beta-elimination of beta-chloro-L-alanine is the release of pyruvate, the rate-determining step for the mutant in the same reaction is the formation of alpha-aminoacrylate. Altogether, these results provide new insights into the catalytic mechanism of cystalysin and highlight the functional role of Tyr 64.