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This Special Issue focusses on monoamine neurotransmitters responsible for mediating neuronal transmission [...]

Aromatic l-amino acid decarboxylase (AADC) deficiency is a complex inherited neurological disorder of monoamine synthesis which results in dopamine and serotonin deficiency. The majority of affected individuals have variable, though often severe cognitive and motor delay, with a complex movement disorder and high risk of premature mortality. For most, standard pharmacological treatment provides only limited clinical benefit. Promising gene therapy approaches are emerging, though may not be either suitable or easily accessible for all patients. To characterize the underlying disease pathophysiology and guide precision therapies, we generated a patient-derived midbrain dopaminergic neuronal model of AADC deficiency from induced pluripotent stem cells. The neuronal model recapitulates key disease features, including absent AADC enzyme activity and dysregulated dopamine metabolism. We observed developmental defects affecting synaptic maturation and neuronal electrical properties, which were improved by lentiviral gene therapy. Bioinformatic and biochemical analyses on recombinant AADC predicted that the activity of one variant could be improved by l-3,4-dihydroxyphenylalanine (l-DOPA) administration; this hypothesis was corroborated in the patient-derived neuronal model, where l-DOPA treatment leads to amelioration of dopamine metabolites. Our study has shown that patient-derived disease modelling provides further insight into the neurodevelopmental sequelae of AADC deficiency, as well as a robust platform to investigate and develop personalized therapeutic approaches., Rossignoli et al. develop the first humanized neuronal model of AADC deficiency. They use this patient-derived neuronal system to elucidate disease mechanisms, and in particular to better define neurodevelopmental features, as well as to test precision therapy approaches.

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease due to mutations in the ddc gene producing AADC, a homodimeric pyridoxal 5′-phosphate-dependent enzyme. The disorder is often fatal in the first decade and is characterized by profound motor impairments and developmental delay. In the last two years, there has been a net rise in the number of patients and variants identified, maybe also pushed by the ongoing gene therapy trials. The majority of the identified genotypes are compound heterozygous (about 70%). Efforts are underway to reach early diagnosis, find possible new markers/new fast methods, and predict clinical outcome. However, no clear correlation of genotype-to-phenotype exists to date. Nevertheless, for homozygous patients, reliable results have been obtained using genetic methods combined with available computational tools on crystal structures corroborated by biochemical investigations on recombinant homodimeric AADC variants that have been obtained and characterized in solution. For these variants, the molecular basis for the defect has been suggested and validated, since it correlates quite well with mildness/severity of the homozygous phenotype. Instead, prediction for compound heterozygous patients is more difficult since complementation effects could happen. Here, by analyzing the existing literature on compound heterozygosity in AADC deficiency and other genetic disorders, we highlight that, in order to assess pathogenicity, the measurement of activity of the AADC heterodimeric variant should be integrated by bioinformatic, structural, and functional data on the whole protein constellation theoretically present in such patients. A wider discussion on symptomatic heterozygosity in AADC deficiency is also presented.

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.

Aromatic- l -amino acid decarboxylase (AADC) deficiency is an ultra-rare inherited autosomal recessive disorder characterized by sharply reduced synthesis of dopamine as well as other neurotransmitters. Symptoms, including hypotonia and movement disorders (especially oculogyric crisis and dystonia) as well as autonomic dysfunction and behavioral disorders, vary extensively and typically emerge in the first months of life. However, diagnosis is difficult, requiring analysis of metabolites in cerebrospinal fluid, assessment of plasma AADC activity, and/or DNA sequence analysis, and is frequently delayed for years. New metabolomics techniques promise early diagnosis of AADC deficiency by detection of 3-O-methyl-dopa in serum or dried blood spots. A total of 82 dopa decarboxylase (DDC) variants in the DDC gene leading to AADC deficiency have been identified and catalogued for all known patients (n = 123). Biochemical and bioinformatics studies provided insight into the impact of many variants. c.714+4A>T, p.S250F, p.R347Q, and p.G102S are the most frequent variants (cumulative allele frequency = 57%), and c.[714+4A>T];[714+4A>T], p.[S250F];[S250F], and p.[G102S];[G102S] are the most frequent genotypes (cumulative genotype frequency = 40%). Known or predicted molecular effect was defined for 79 variants. Most patients experience an unrelenting disease course with poor or no response to conventional medical treatments, including dopamine agonists, monoamine oxidase inhibitors, and pyridoxine derivatives. The advent of gene therapy represents a potentially promising new avenue for treatment of patients with AADC deficiency. Clinical studies based on the direct infusion of engineered adeno-associated virus type 2 vectors into the putamen have demonstrated acceptable safety and tolerability and encouraging improvement in motor milestones and cognitive symptoms. The success of gene therapy in AADC deficiency treatment will depend on timely diagnosis to facilitate treatment administration before the onset of neurologic damage.

Compound heterozygosis is the most diffuse and hardly to tackle condition in aromatic amino acid decarboxylase (AADC) deficiency, a genetic disease leading to severe neurological impairment. Here, by using an appropriate vector, we succeeded in obtaining high yields of AADC protein and characterizing two new heterodimers, T69M/S147R and C281W/M362T, detected in two AADC deficiency patients. We performed an extensive biochemical characterization of the heterodimeric recombinant proteins and of the related homodimers, by a combination of dichroic and fluorescence spectroscopy and activity assays together with bioinformatic analyses. We found that T69M/S147R exhibits negative complementation in terms of activity but it is more stable than the average of the homodimeric counterparts. The heterodimer C281W/M362T retains a nearly good catalytic efficiency, whereas M362T homodimer is less affected and C281W homodimer is recovered as insoluble. These results, which are consistent with the related phenotypes, and the data emerging from previous studies, suggest that the severity of AADC deficiency is not directly explained by positive or negative complementation phenomena, but rather depends on: i) the integrity of one or both active sites; ii) the structural and functional properties of the entire pool of AADC proteins expressed. Overall, this integrated and cross-sectional approach enables proper characterization and depicts the functional result of subunit interactions in the dimeric structure and will help to elucidate the physio-pathological mechanisms in AADC deficiency.

Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells.

Non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana are hexacoordinate heme-proteins that likely have different biological roles, in view of diverse tissue localization, expression pattern, and ligand binding properties. Herein, we expand upon previous biophysical studies on these isoforms, focusing on their oligomeric states and circular dichroism (CD) characteristics. We found that AHb1 exists in solution in a concentration-dependent monomer-dimer equilibrium, while AHb2 is present only as a monomer. The quaternary structure of AHb1 affects its degree of hexacoordination with the formation of the dimer that enhances pentacoordination. Accordingly, the mutant of a conserved residue within the dimeric interface, AHb1-T45A, which is mostly monomeric in solution, has an equilibrium that is shifted toward a hexacoordinate form compared to the wild-type protein. CD studies further support differences in the globin&rsquo, s structure and heme moiety. The Soret CD spectra for AHb2 are opposite in sense to those for AHb1, reflecting different patterns of heme-protein side chain contacts in the two proteins. Moreover, the smaller contribution of the heme to the near-UV CD in AHb2 compared to AHb1 suggests a weaker heme-protein association in AHb2. Our data corroborate the structural diversity of AHb1 and AHb2 and confirm the leghemoglobin-like structural properties of AHb2.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter γ-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including γ-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.

Some coronavirus disease 2019 (COVID-19) patients develop acute pneumonia which can result in a cytokine storm syndrome in response to Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infection. The most effective anti-inflammatory drugs employed so far in severe COVID-19 belong to the cytokine-directed biological agents, widely used in the management of many autoimmune diseases. In this paper we analyze the efficacy of epigallocatechin 3-gallate (EGCG), the most abundant ingredient in green tea leaves and a well-known antioxidant, in counteracting autoimmune diseases, which are dominated by a massive cytokines production. Indeed, many studies registered that EGCG inhibits signal transducer and activator of transcription (STAT)1/3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factors, whose activities are crucial in a multiplicity of downstream pro-inflammatory signaling pathways. Importantly, the safety of EGCG/green tea extract supplementation is well documented in many clinical trials, as discussed in this review. Since EGCG can restore the natural immunological homeostasis in many different autoimmune diseases, we propose here a supplementation therapy with EGCG in COVID-19 patients. Besides some antiviral and anti-sepsis actions, the major EGCG benefits lie in its anti-fibrotic effect and in the ability to simultaneously downregulate expression and signaling of many inflammatory mediators. In conclusion, EGCG can be considered a potential safe natural supplement to counteract hyper-inflammation growing in COVID-19.

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.

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

Pyridoxal 5'-phosphate (PLP)-dependent enzymes catalyze a wide range of reactions of amino acids and amines, with the exception of glycogen phosphorylase which exhibits peculiar both substrate preference and chemical mechanism. They represent about 4% of the gene products in eukaryotic cells. Although structure-function investigations regarding these enzymes are copious, their regulation by post-translational modifications is largely unknown. Protein phosphorylation is the most common post-translational modification fundamental in mediating diverse cellular functions. This review aims at summarizing the current knowledge on regulation of PLP enzymes by phosphorylation. Starting from the paradigmatic PLP-dependent glycogen phosphorylase, the first phosphoprotein discovered, we collect data in literature regarding functional phosphorylation events of eleven PLP enzymes belonging to different fold types and discuss the impact of the modification in affecting their activity and localization as well as the implications on the pathogenesis of diseases in which many of these enzymes are involved. The pivotal question is to correlate the structural consequences of phosphorylation among PLP enzymes of different folds with the functional modifications exerted in terms of activity or conformational changes or others. Although the literature shows that the phosphorylation of PLP enzymes plays important roles in mediating diverse cellular functions, our recapitulation of clue findings in the field makes clear that there is still much to be learnt. Besides mass spectrometry-based proteomic analyses, further biochemical and structural studies on purified native proteins are imperative to fully understand and predict how phosphorylation regulates PLP enzymes and to find the relationship between addition of a phosphate moiety and physiological response.

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

Aromatic amino acid decarboxylase (AADC) deficiency is a rare autosomal neurometabolic disorder caused by a deficit of AADC, a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which catalyzes the synthesis of dopamine and serotonin. While many studies have highlighted the molecular defects of the homozygous pathogenic variants, so far only a study investigated heterozygous variants at protein level. Here, we report a clinical case of one AADC deficiency compound heterozygous patient bearing the A91V mutation and the novel C410G mutation. To elucidate its enzymatic phenotype, the A91V and C410G homodimers were first expressed in Escherichia coli, purified and characterized. Although both apo variants display an unaltered overall tertiary structure, they show a 20-fold decreased PLP binding affinity. The C410G mutation only causes a 4-fold decrease of the catalytic efficiency, while the A91V mutation causes a 1300-fold decrease of the kcat/Km, and changes in the holoAADC consisting in a marked alteration of the tertiary structure and the coenzyme microenvironment. Structural analyses of these mutations are in agreement with these data. Unfortunately, the C410G/A91V heterodimer was constructed, expressed and purified in rather modest amount. Anyway, measurements of decarboxylase activity indicate that its putative kcat value is lower than that predicted by averaging the kcat values of the two parental enzymes. This indicates a negative interallelic complementation between the C410G and A91V monomers. Overall, this study allowed to relate the clinical to the enzymatic phenotype of the patient and to extend knowledge in the clinical and molecular pathogenesis of AADC deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter &gamma, amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including &gamma, &ndash, hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T &gt, C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.

Histidine decarboxylase is a pyridoxal 5'-phosphate enzyme catalyzing the conversion of histidine to histamine, a bioactive molecule exerting its role in many modulatory processes. The human enzyme is involved in many physiological functions, such as neurotransmission, gastrointestinal track function, cell growth, and differentiation. Here, we studied the functional properties of the human enzyme and, in particular, the effects exerted at the protein level by two cysteine residues: Cys-180 and Cys-418. Surprisingly, the enzyme exists in an equilibrium between a reduced and an oxidized form whose extent depends on the redox state of Cys-180. Moreover, we determined that (i) the two enzymatic redox species exhibit modest structural changes in the coenzyme microenvironment and (ii) the oxidized form is slightly more active and stable than the reduced one. These data are consistent with the model proposed by bioinformatics analyses and molecular dynamics simulations in which the Cys-180 redox state could be responsible for a structural transition affecting the C-terminal domain reorientation leading to active site alterations. Furthermore, the biochemical properties of the purified C180S and C418S variants reveal that C180S behaves like the reduced form of the wild-type enzyme, while C418S is sensitive to reductants like the wild-type enzyme, thus allowing the identification of Cys-180 as the redox sensitive switch. On the other hand, Cys-418 appears to be a residue involved in aggregation propensity. A possible role for Cys-180 as a regulatory switch in response to different cellular redox conditions could be suggested.

Aromatic amino acid, cysteine sulfinic acid, glutamate and histidine decarboxylases, belonging to group II of pyridoxal 5'-phosphate-dependent enzymes, catalyze the synthesis of dopamine/serotonin, hypotaurine, γ-aminobutyric acid and histamine, respectively. Considering that these reaction products are all essential bioactive molecules, group II decarboxylases have been long studied from an evolutionary, biochemical and pharmacological standpoint. Despite the fact that they all belong to a common fold-type, during evolution each decarboxylase has evolved unique structural elements responsible for its substrate specificity. Combining a literature update with bioinformatic analyses, this review focuses on some structural determinants shared by these enzymes revealing their intrinsic substrate specificity and highlighting the importance of some residues/regions for catalytic competence. In particular, two key structural features emerge: 1) a mobile catalytic loop, and 2) an open-to-close conformation accompanying the apo-holo transition. Drawing attention on these elements is crucial in correlating subtle structural modifications to functional properties for the understanding, at a molecular level of a pathological condition. This is corroborated by the increasingly important role played by these decarboxylases in several different pathological states (autoimmune diseases, type I diabetes, Parkinson's disease, aromatic amino acid decarboxylase deficiency, Tourette's syndrome and cholangiocarcinoma).

Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2(-/-) mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2(-/-) red cells and reduced Prx2(-/-) red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2(-/-) mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2(-/-) mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2(-/-) mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.

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.

Resveratrol, a polyphenolic-stilbene, has received increased attention in the last decade due to its wide range of biological activities. Beta(β)-thalassemias are inherited red cell disorders, found worldwide, characterized by ineffective erythropoiesis and red cell oxidative damage with reduced survival. We evaluated the effects of low-dose-resveratrol (5 μM) on in vitro human erythroid differentiation of CD34(+) from normal and β-thalassemic subjects. We found that resveratrol induces accelerated erythroid-maturation, resulting in the reduction of colony-forming units of erythroid cells and increased intermediate and late erythroblasts. In sorted colony-forming units of erythroid cells resveratrol activates Forkhead-box-class-O3, decreases Akt activity and up-regulates anti-oxidant enzymes as catalase. In an in vivo murine model for β-thalassemia, resveratrol (2.4 mg/kg) reduces ineffective erythropoiesis, increases hemoglobin levels, reduces reticulocyte count and ameliorates red cell survival. In both wild-type and β-thalassemic mice, resveratrol up-regulates scavenging enzymes such as catalase and peroxiredoxin-2 through Forkhead-box-class-O3 activation. These data indicate that resveratrol inhibits Akt resulting in FoxO3 activation with upregulation of cytoprotective systems enabling the pathological erythroid precursors to resist the oxidative damage and continue to differentiate. Our data suggest that the dual effect of resveratrol on erythropoiesis through activation of FoxO3 transcriptional factor combined with the amelioration of oxidative stress in circulating red cells may be considered as a potential novel therapeutic strategy in treating β-thalassemia.

β-thalassemia is a worldwide distributed monogenic red cell disorder, characterized by the absence or reducedβ-globin chain synthesis. Despite the extensive knowledge of the molecular defects causingβ-thalassemia, less is known about the mechanisms responsible for the associated ineffective erythropoiesis and reduced red cell survival, which sustain anemia ofβ-thalassemia. The unbalance of alpha-gamma chain and the presence of pathological free iron promote a severe red cell membrane oxidative stress, which results in abnormalβ-thalassemic red cell features. These cells are precociously removed by the macrophage system through two mechanisms: the removal of phosphatidylserine positive cells and through the natural occurring antibody produced against the abnormally clustered membrane protein band 3. In the present review we will discuss the changes inβ-thalassemic red cell homeostasis related to the oxidative stress and its connection with production of microparticles and with malaria infection. The reactive oxygen species (ROS) are also involved in ineffective erythropoiesis ofβ-thalassemia through still partially known pathways. Novel cytoprotective systems such as ASHP, eIF2α, and peroxiredoxin-2 have been suggested to be important against ROS inβ-thalassemic erythropoiesis. Finally, we will discuss the results of the majorin vitroandin vivostudies with antioxidants inβ-thalassemia.

Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyn-containing high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD34+-derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in chorea-acanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation.

Since their discovery in the mid-nineties, peroxiredoxins have drawn much attention and the number of papers publications on different Prxs has been multiplied. The rise in interest in this topic is probably due, at least in part, to the large and further increasing functions attributed to the members of this family of ubiquitous proteins, including many redox and non-redox physiological functions. This review presents a Since their discovery in the mid-nineties, peroxiredoxins have drawn much attention and the number of publications on different Prxs has been multiplied. The rise in interest in this topic is probably due, at least in part, to the large and further increasing functions attributed to the members of this family of ubiquitous proteins, including many redox and non-redox physiological functions. This review presents a literature survey of the protein partners of the human Peroxiredoxin-1 and Peroxiredoxin- 2 of the Peroxiredoxin 1 subfamily, the most abundant class. Three sequence motifs, or combinations thereof, were found in the protein partners, namely, CXXC, PXXP, and LXXLL. These findings are discussed in light of i) protein partner localization, function and biological pathways and ii) the peroxiredoxins regions important for partner interaction, as revealed by the Peroxiredoxin-1-Sulfiredoxin-1 complex structure. The outcome of these analyses is expected to unravel some common molecular bases underlying peroxiredoxins propensity to bind a partner, as well as to propose a functional role for this interaction that could help to widen the biological role of this important class of enzymes.

We report here a clinical case of a patient with a novel mutation (Arg347→Gly) in the gene encoding aromatic amino acid decarboxylase (AADC) that is associated with AADC deficiency. The variant R347G in the purified recombinant form exhibits, similarly to the pathogenic mutation R347Q previously studied, a 475-fold drop of kcat compared to the wild-type enzyme. In attempting to unravel the reason(s) for this catalytic defect, we have carried out bioinformatics analyses of the crystal structure of AADC-carbidopa complex with the modelled catalytic loop (residues 328-339). Arg347 appears to interact with Phe103, as well as with both Leu333 and Asp345. We have then prepared and characterized the artificial F103L, R347K and D345A mutants. F103L, D345A and R347K exhibit about 13-, 97-, and 345-fold kcat decrease compared to the wild-type AADC, respectively. However, unlike F103L, the R347G, R347K and R347Q mutants as well as the D345A variant appear to be more defective in catalysis than in protein folding. Moreover, the latter mutants, unlike the wild-type protein and the F103L variant, share a peculiar binding mode of dopa methyl ester consisting of formation of a quinonoid intermediate. This finding strongly suggests that their catalytic defects are mainly due to a misplacement of the substrate at the active site. Taken together, our results highlight the importance of the Arg347-Leu333-Asp345 hydrogen-bonds network in the catalysis of AADC and reveal the molecular basis for the pathogenicity of the variants R347. Following the above results, a therapeutic treatment for patients bearing the mutation R347G is proposed.

Backround: Parkinson’s disease is a pathology involving the progressive degeneration of dopaminergic neurons in the substantia nigra of the brain. L-DOPA combined with an inhibitor of DOPA decarboxylase, a pyridoxal 5’-phosphate-dependent enzyme, is still the most effective treatment for symptoms of Parkinson's disease. LDOPA increases synaptic dopamine, while the inhibitor of peripheral DOPA decarboxylase reduces the conversion of L-DOPA to dopamine in the systemic circulation, allowing for greater L-DOPA distribution into the central nervous system. CarbiDOPA and benserazide are the inhibitors currently used in Parkinson's disease treatment. However, carbiDOPA and trihydroxybenzylhydrazine, the active metabolite of benserazide, are substrate analogues both endowed with a hydrazine function, which irreversibly bind not only to DDC but also to free pyridoxal 5’-phosphate and pyridoxal 5’-phosphate-dependent enzymes. Therefore, the lack of DOPA decarboxylase specificity, responsible for various side effects and adverse reactions, is a negative factor in such treatment of the disease. Results and Conclusion: Aim of this review is to report on the most recent investigations regarding new DOPA decarboxylase inhibitors that could represent the starting point for possible Parkinson's disease drugs development. We focused on the common chemical features among all the identified inhibitors in order to seek shared structural motifs that could be involved in inhibition. Then, we highlighted the extent of inhibition, measured by means of in vitro and/or cell-based assays. Finally, we pointed out the state of the art in the metabolism of such classes of compounds, and discussed the possible advances in Parkinson's disease pharmacological treatment.

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.

Acanthocytic RBCs are a peculiar diagnostic feature of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder. Although recent years have witnessed some progress in the molecular characterization of ChAc, the mechanism(s) responsible for generation of acanthocytes in ChAc is largely unknown. As the membrane protein composition of ChAc RBCs is similar to that of normal RBCs, we evaluated the tyrosine (Tyr)–phosphorylation profile of RBCs using comparative proteomics. Increased Tyr phosphorylation state of several membrane proteins, including band 3, β-spectrin, and adducin, was noted in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs, band 3 Tyr phosphorylation by Lyn was independent of the canonical Syk-mediated pathway. The ChAc-associated alterations in RBC membrane protein organization appear to be the result of increased Tyr phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton as supported by coimmunoprecipitation of β-adducin with band 3 only in ChAc RBC-membrane treated with the Lyn-inhibitor PP2. We propose this altered association between membrane skeleton and membrane proteins as novel mechanism in the generation of acanthocytes in ChAc.

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.

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.

We have determined the kinetic parameters for Dopa decarboxylase (DDC) of three ring-fluorinated analogs of 3,4-dihydroxyphenylalanine (Dopa). The rank order of catalytic efficiency of decarboxylation (k(cat)/K(m)) is Dopa>6-F-Dopa>2-F-Dopa>5-F-Dopa. This rank is consistent with previous in vivo and in vitro studies which indicate that, of the fluorinated analogs, 6-F-Dopa has pharmacokinetics that are most suited for positron emission tomographic (PET) evaluation of dopamine function. The effectiveness of PET as a diagnostic tool, the convenient half-life of (18)F (110 min) and the favorable pharmacokinetics of 6-[(18)F]FDOPA have combined to make this an extremely valuable reagent to study dopaminergic activity. The reactions of the related fluorinated DOPS analogs show that, while 6-F-threo-3,4-(dihydroxyphenyl)serine (DOPS) is decarboxylated at approximately the same rate as the non-fluorinated substrate, 2-F-threo-DOPS is not converted into the corresponding amine. In both cases a Pictet-Spengler condensation with the pyridoxal 5(')-phosphate (PLP) cofactor occurs to produce tetrahydroisoquinolines. Condensation of fluorinated catecholamines and catechol amino acids with endogenous aldehydes will be investigated as an approach to study possible mechanisms of L-Dopa-linked neurotoxicity.

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) • poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) • poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.

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 (k(inact)/K(i)) 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.

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.

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, Kd, of the enzyme for pyridoxal 5ƀ-phosphate (PLP). Instead, at enzyme concentrations higher than Kd the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5ƀ-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 Kd, 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 O2. 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 O2 control. In contrast, the reactivity of the enzyme with D-aromatic amino acids does not change in the presence or absence of O2. These and other results, together with previous results on the effect exerted by O2 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 O2 on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines.

Ornithine decarboxylase (ODC) from Lactobacillus 30a catalyses the cleavage of alpha-methylornithine into ammonia and 2-methyl-1-pyrroline; glutamate decarboxylase (GAD) from Escherichia coli catalyses the cleavage of alpha-methylglutamate into ammonia and laevulinic acid. In our analyses, 2-methyl-1-pyrroline and laevulinic acid were identified by HPLC and mass spectroscopic analysis, and ammonia was identified by means of glutamate dehydrogenase. Molecular oxygen was consumed during these reactions in a 1:2 molar ratio with respect to the products. The catalytic efficiencies (k(cat)/K(m)) of the reactions catalysed by ODC and GAD were determined as 12500 and 9163 M(-1).min(-1) respectively. When the reactions were performed under anaerobic conditions, no ammonia, 2-methyl-1-pyrroline or laevulinic acid was produced to a significant extent. The formation of ammonia and O(2) consumption (in a 1:2 molar ratio with respect to ammonia) were also detected during the reaction of ODC and GAD with putrescine and gamma-aminobutyrate respectively. Taken together, these findings clearly indicate that ODC and GAD catalyse an oxidative deamination of their decarboxylation products, a reaction similar to that catalysed by dopa decarboxylase (DDC) with alpha-methyldopa [Bertoldi, Dominici, Moore, Maras and Borri Voltattorni (1998) Biochemistry 37, 6552-6561]. Furthermore, this reaction was accompanied by a decarboxylation-dependent transamination occurring for GAD, DDC and ODC with a frequency of approx. 0.24%, 1% and 9% respectively compared with that of oxidative deamination.

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.

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111S mutant enzymes exhibit Kcat values of about 50% and 15%, respectively, at pH 6.8, while the K(m) values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (L-5-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-111-->Ala mutant displays a reduced affinity for pyridoxal 5'-phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild-type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420-->390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Borri Voltattorni C. 1991. Eur J Biochem 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.

Band-3 (B3), the anion transporter is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In the present study, using a variety of experimental approaches including crosslinking studies, fluorescence and dichroic measurements, surface-plasmon-resonance analysis, and proteolytic digestion assays, we document that the anti-oxidant protein, Peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine (PHZ) of RBCs prevented membrane association of PRDX2 implying over lapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acid of this domain are crucial for PRDX2 membrane-association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function.