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1.
Int J Mol Sci ; 24(4)2023 Feb 08.
Article in English | MEDLINE | ID: mdl-36834788

ABSTRACT

Deficit of human ornithine aminotransferase (hOAT), a mitochondrial tetrameric pyridoxal-5'-phosphate (PLP) enzyme, leads to gyrate atrophy of the choroid and retina (GA). Although 70 pathogenic mutations have been identified, only few enzymatic phenotypes are known. Here, we report biochemical and bioinformatic analyses of the G51D, G121D, R154L, Y158S, T181M, and P199Q pathogenic variants involving residues located at the monomer-monomer interface. All mutations cause a shift toward a dimeric structure, and changes in tertiary structure, thermal stability, and PLP microenvironment. The impact on these features is less pronounced for the mutations of Gly51 and Gly121 mapping to the N-terminal segment of the enzyme than those of Arg154, Tyr158, Thr181, and Pro199 belonging to the large domain. These data, together with the predicted ΔΔG values of monomer-monomer binding for the variants, suggest that the proper monomer-monomer interactions seem to be correlated with the thermal stability, the PLP binding site and the tetrameric structure of hOAT. The different impact of these mutations on the catalytic activity was also reported and discussed on the basis of the computational information. Together, these results allow the identification of the molecular defects of these variants, thus extending the knowledge of enzymatic phenotypes of GA patients.


Subject(s)
Gyrate Atrophy , Ornithine-Oxo-Acid Transaminase , Humans , Atrophy/pathology , Choroid/metabolism , Gyrate Atrophy/genetics , Mutation , Ornithine , Ornithine-Oxo-Acid Transaminase/metabolism , Pyridoxal Phosphate , Retina/metabolism
2.
Int J Mol Sci ; 22(6)2021 Mar 19.
Article in English | MEDLINE | ID: mdl-33808712

ABSTRACT

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


Subject(s)
Amino Acid Metabolism, Inborn Errors/etiology , Amino Acid Metabolism, Inborn Errors/metabolism , Aromatic-L-Amino-Acid Decarboxylases/deficiency , Disease Susceptibility , Aromatic-L-Amino-Acid Decarboxylases/chemistry , Aromatic-L-Amino-Acid Decarboxylases/genetics , Aromatic-L-Amino-Acid Decarboxylases/metabolism , Biomarkers , Catalysis , Dopamine/metabolism , Homozygote , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Interaction Domains and Motifs , Serotonin/metabolism , Structure-Activity Relationship
3.
Biochim Biophys Acta Proteins Proteom ; 1869(1): 140555, 2021 01.
Article in English | MEDLINE | ID: mdl-33068755

ABSTRACT

Gyrate Atrophy (GA) of the choroid and retina (MIM# 258870) is an autosomal recessive disorder due to mutations of the OAT gene encoding ornithine-delta-aminotransferase (OAT), associated with progressive retinal deterioration and blindness. The disease has a theoretical global incidence of approximately 1:1,500,000. OAT is mainly involved in ornithine catabolism in adults, thus explaining the hyperornithinemia as hallmark of the disease. Patients are treated with an arginine-restricted diet, to limit ornithine load, or the administration of Vitamin B6, a precursor of the OAT coenzyme pyridoxal phosphate. Although the clinical and genetic aspects of GA are known for many years, the enzymatic phenotype of pathogenic variants and their response to Vitamin B6, as well as the molecular mechanisms explaining retinal damage, are poorly clarified. Herein, we provide an overview of the current knowledge on the biochemical properties of human OAT and on the molecular, cellular, and clinical aspects of GA.


Subject(s)
Coenzymes/administration & dosage , Gyrate Atrophy/diet therapy , Gyrate Atrophy/enzymology , Ornithine-Oxo-Acid Transaminase/deficiency , Pyridoxal Phosphate/administration & dosage , Vitamin B 6/administration & dosage , Arginine/metabolism , Choroid/enzymology , Choroid/pathology , Chromosomes, Human, Pair 10 , Diet/methods , Gene Expression , Gyrate Atrophy/genetics , Gyrate Atrophy/pathology , Humans , Models, Molecular , Mutation , Ornithine/metabolism , Ornithine-Oxo-Acid Transaminase/chemistry , Ornithine-Oxo-Acid Transaminase/genetics , Protein Multimerization , Protein Structure, Secondary , Retina/enzymology , Retina/pathology
4.
Mol Genet Metab ; 127(2): 132-137, 2019 06.
Article in English | MEDLINE | ID: mdl-31104889

ABSTRACT

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 Ì´ 20-fold decreased PLP binding affinity. The C410G mutation only causes a Ì´ 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.


Subject(s)
Amino Acid Metabolism, Inborn Errors/genetics , Aromatic-L-Amino-Acid Decarboxylases/deficiency , Aromatic-L-Amino-Acid Decarboxylases/genetics , Mutation , Aromatic-L-Amino-Acid Decarboxylases/metabolism , Child, Preschool , Dopamine/metabolism , Genotype , Heterozygote , Humans , Male , Serotonin/metabolism
5.
FEBS J ; 286(14): 2787-2798, 2019 07.
Article in English | MEDLINE | ID: mdl-30957963

ABSTRACT

Among the over 50 gyrate atrophy-causing mutations of ornithine δ-aminotransferase (OAT), the R180T involves an active site residue located at the dimer interface, which in the crystal structure of OAT complexed with 5-fluoromethylornithine engages a salt bridge with the α-carboxylate of the substrate analogue. Starting from the previous finding that no transaminase activity was detected in CHO-K1 cells expressing the R180T variant, here we try to shed light at the protein level on the structural and/or functional defects of the R180T variant. To this aim, the variant has been cloned, expressed, purified and characterized by a combination of biochemical and structural studies. Although the R180T variant shares a similar overall conformation with the wild-type, its crystal structure solved at 1.8 Çº reveals slight structural alterations at the active site and at the dimeric interface. These changes are consistent with the spectroscopic and kinetic results, indicating that the variant, as compared with the wild-type OAT, shows (a) an increased Km value for l-ornithine (l-Orn), (b) an altered pyridoxal 5'-phosphate binding mode and affinity and (c) an increased thermostability. In addition, the R180T mutant exhibits a remarkable loss of catalytic activity and is endowed with the ability to catalyse not only the δ-transamination but also, albeit to a lesser extent, the α-transamination of l-Orn. Overall, these data indicate that the slight structural changes caused by the R180T mutation, preventing a proper collocation of l-Orn at the active site of OAT, are responsible for the notable reduction of the catalytic efficiency. ENZYMES: Ornithine aminotransferase EC 2.6.1.13. DATABASES: 6HX7.pdb.


Subject(s)
Gyrate Atrophy/genetics , Ornithine-Oxo-Acid Transaminase/genetics , Biocatalysis , Crystallography, X-Ray , Enzyme Stability , Humans , Kinetics , Magnetic Resonance Spectroscopy , Molecular Docking Simulation , Mutation , Ornithine-Oxo-Acid Transaminase/chemistry
6.
Biochemistry ; 57(44): 6336-6348, 2018 11 06.
Article in English | MEDLINE | ID: mdl-30346159

ABSTRACT

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.


Subject(s)
Cysteine/chemistry , Histidine Decarboxylase/chemistry , Histidine Decarboxylase/metabolism , Mutation , Pyridoxal Phosphate/metabolism , Amino Acid Sequence , Catalysis , Catalytic Domain , Crystallography, X-Ray , Cysteine/genetics , Cysteine/metabolism , Histidine Decarboxylase/genetics , Humans , Models, Molecular , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Conformation , Sequence Homology
7.
Biochim Biophys Acta Mol Basis Dis ; 1864(11): 3629-3638, 2018 11.
Article in English | MEDLINE | ID: mdl-30251682

ABSTRACT

Gyrate atrophy (GA) is a rare recessive disorder characterized by progressive blindness, chorioretinal degeneration and systemic hyperornithinemia. GA is caused by point mutations in the gene encoding ornithine δ-aminotransferase (OAT), a tetrameric pyridoxal 5'-phosphate-dependent enzyme catalysing the transamination of l-ornithine and α-ketoglutarate to glutamic-γ-semialdehyde and l-glutamate in mitochondria. More than 50 OAT variants have been identified, but their molecular and cellular properties are mostly unknown. A subset of patients is responsive to pyridoxine administration, although the mechanisms underlying responsiveness have not been clarified. Herein, we studied the effects of the V332M mutation identified in pyridoxine-responsive patients. The Val332-to-Met substitution does not significantly affect the spectroscopic and kinetic properties of OAT, but during catalysis it makes the protein prone to convert into the apo-form, which undergoes unfolding and aggregation under physiological conditions. By using the CRISPR/Cas9 technology we generated a new cellular model of GA based on HEK293 cells knock-out for the OAT gene (HEK-OAT_KO). When overexpressed in HEK-OAT_KO cells, the V332M variant is present in an inactive apodimeric form, but partly shifts to the catalytically-competent holotetrameric form in the presence of exogenous PLP, thus explaining the responsiveness of these patients to pyridoxine administration. Overall, our data represent the first integrated molecular and cellular analysis of the effects of a pathogenic mutation in OAT. In addition, we validated a novel cellular model for the disease that could prove instrumental to define the molecular defect of other GA-causing variants, as well as their responsiveness to pyridoxine and other putative drugs.


Subject(s)
Gyrate Atrophy/genetics , Ornithine-Oxo-Acid Transaminase/genetics , Protein Aggregation, Pathological/genetics , Pyridoxal Phosphate/metabolism , Vitamin B Complex/pharmacology , CRISPR-Cas Systems/genetics , Coenzymes/metabolism , Enzyme Assays , Gene Knockout Techniques , Gyrate Atrophy/drug therapy , Gyrate Atrophy/pathology , HEK293 Cells , Holoenzymes/genetics , Holoenzymes/metabolism , Humans , Mutagenesis, Site-Directed , Ornithine-Oxo-Acid Transaminase/metabolism , Point Mutation , Protein Aggregation, Pathological/drug therapy , Protein Aggregation, Pathological/pathology , Pyridoxine/pharmacology , Pyridoxine/therapeutic use , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Treatment Outcome , Vitamin B Complex/therapeutic use
8.
IUBMB Life ; 70(3): 215-223, 2018 03.
Article in English | MEDLINE | ID: mdl-29356298

ABSTRACT

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.


Subject(s)
Amino Acid Metabolism, Inborn Errors/embryology , Aromatic-L-Amino-Acid Decarboxylases/chemistry , Aromatic-L-Amino-Acid Decarboxylases/deficiency , Protein Multimerization/genetics , Recombinant Proteins/chemistry , Amino Acid Metabolism, Inborn Errors/enzymology , Amino Acid Metabolism, Inborn Errors/genetics , Aromatic-L-Amino-Acid Decarboxylases/genetics , Aromatic-L-Amino-Acid Decarboxylases/metabolism , Catalysis , Dopamine/biosynthesis , Heterozygote , Humans , Mutation , Protein Folding , Recombinant Proteins/genetics , Serotonin/biosynthesis
9.
Handb Exp Pharmacol ; 245: 313-343, 2018.
Article in English | MEDLINE | ID: mdl-29071511

ABSTRACT

Protein misfolding is becoming one of the main mechanisms underlying inherited enzymatic deficits. This review is focused on primary hyperoxalurias, a group of disorders of glyoxylate detoxification associated with massive calcium oxalate deposition mainly in the kidneys. The most common and severe form, primary hyperoxaluria Type I, is due to the deficit of liver peroxisomal alanine/glyoxylate aminotransferase (AGT). Various studies performed in the last decade clearly evidence that many pathogenic missense mutations prevent the AGT correct folding, leading to various downstream effects including aggregation, increased degradation or mistargeting to mitochondria. Primary hyperoxaluria Type II and primary hyperoxaluria Type III are due to the deficit of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) and 4-hydroxy-2-oxoglutarate aldolase (HOGA1), respectively. Although the molecular features of pathogenic variants of GRHPR and HOGA1 have not been investigated in detail, the data available suggest that some of them display folding defects. Thus, primary hyperoxalurias can be ranked among protein misfolding disorders, because in most cases the enzymatic deficit is due to the inability of each enzyme to reach its native and functional conformation. It follows that molecules able to improve the folding yield of the enzymes involved in each disease form could represent new therapeutic strategies.


Subject(s)
Hyperoxaluria, Primary/etiology , Proteostasis Deficiencies/etiology , Animals , Humans , Hydroxypyruvate Reductase/genetics , Molecular Chaperones/therapeutic use , Oxo-Acid-Lyases/genetics , Protein Folding , Transaminases/chemistry , Transaminases/genetics
10.
Protein J ; 36(3): 174-185, 2017 06.
Article in English | MEDLINE | ID: mdl-28345116

ABSTRACT

Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5'-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent Tm values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer-dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, Tm values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.


Subject(s)
Ornithine-Oxo-Acid Transaminase/chemistry , Protein Multimerization , Amino Acid Substitution , Apoenzymes/chemistry , Apoenzymes/genetics , Enzyme Stability , Holoenzymes/chemistry , Holoenzymes/genetics , Hot Temperature , Humans , Mutation, Missense , Ornithine-Oxo-Acid Transaminase/genetics , Protein Structure, Quaternary
11.
Biochimie ; 131: 137-148, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27720751

ABSTRACT

In this work the dimerization process of the minor allelic form of human alanine glyoxylate aminotransferase, a pyridoxal 5'-phosphate enzyme, was investigated. Bioinformatic analyses followed by site-directed mutagenesis, size exclusion chromatography and catalytic activity experiments allowed us to identify Arg118, Phe238 and Phe240 as interfacial residues not essential for transaminase activity but important for dimer-monomer dissociation. The apo and the holo forms of the triple mutant R118A-Mi/F238S-Mi/F240S-Mi display a dimer-monomer equilibrium dissociation constant value at least ~260- and 31-fold larger, respectively, than the corresponding ones of AGT-Mi. In the presence of PLP, the apomonomer of the triple mutant undergoes a biphasic process: the fast phase represents the formation of an inactive PLP-bound monomer, while the slow phase depicts the monomer-monomer association that parallels the regain of transaminase activity. The latter events occur with a rate constant of ~0.02 µM-1min-1. In the absence of PLP, the apomonomer is also able to dimerize but with a rate constant value ~2700-fold lower. Thereafter, the possible interference with the dimerization process of AGT-Mi exerted by the mutated residues in the I244T-Mi and F152I-Mi variants associated with Primary Hyperoxaluria type 1 was investigated by molecular dynamics simulations. On the basis of the present and previous studies, a model for the dimerization process of AGT-Mi, I244T-Mi and F152I-Mi, which outlines the structural defects responsible for the complete or partial mistargeting of the pathogenic variants, was proposed and discussed.


Subject(s)
Mutation , Protein Multimerization , Transaminases/chemistry , Transaminases/genetics , Algorithms , Amino Acid Substitution , Circular Dichroism , Humans , Hydrophobic and Hydrophilic Interactions , Kinetics , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Pyridoxal Phosphate/chemistry , Pyridoxal Phosphate/metabolism , Spectrometry, Fluorescence , Transaminases/metabolism
12.
Biochim Biophys Acta ; 1854(10 Pt A): 1280-9, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26149463

ABSTRACT

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.


Subject(s)
Apoenzymes/chemistry , Holoenzymes/chemistry , Mutation , Pyridoxine/pharmacology , Transaminases/chemistry , Alanine/chemistry , Alanine/metabolism , Alleles , Animals , Apoenzymes/genetics , Apoenzymes/metabolism , CHO Cells , Cricetulus , Dose-Response Relationship, Drug , Enzyme Assays , Gene Expression , Glyoxylates/chemistry , Glyoxylates/metabolism , Holoenzymes/genetics , Holoenzymes/metabolism , Humans , Kinetics , Mutagenesis, Site-Directed , Protein Conformation/drug effects , Protein Folding/drug effects , Pyridoxal Phosphate/chemistry , Pyridoxal Phosphate/metabolism , Pyridoxine/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility , Transaminases/genetics , Transaminases/metabolism
13.
Hum Mol Genet ; 23(22): 5998-6007, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-24990153

ABSTRACT

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5'-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients.


Subject(s)
Hyperoxaluria, Primary/enzymology , Hyperoxaluria, Primary/genetics , Mutation, Missense , Transaminases/genetics , Adolescent , Adult , Alleles , Amino Acid Substitution , Female , Heterozygote , Homozygote , Humans , Male , Protein Transport , Transaminases/metabolism
14.
Proc Natl Acad Sci U S A ; 111(25): E2524-9, 2014 Jun 24.
Article in English | MEDLINE | ID: mdl-24927554

ABSTRACT

The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.


Subject(s)
Autoimmunity , Glutamate Decarboxylase , Homeostasis/immunology , Molecular Dynamics Simulation , Neurotransmitter Agents , gamma-Aminobutyric Acid , Autoantibodies/immunology , Diabetes Mellitus, Type 1/immunology , Glutamate Decarboxylase/chemistry , Glutamate Decarboxylase/genetics , Glutamate Decarboxylase/immunology , Humans , Neurotransmitter Agents/chemistry , Neurotransmitter Agents/genetics , Neurotransmitter Agents/immunology , Protein Multimerization , Structure-Activity Relationship , gamma-Aminobutyric Acid/chemistry , gamma-Aminobutyric Acid/genetics , gamma-Aminobutyric Acid/immunology
15.
PLoS One ; 7(2): e31610, 2012.
Article in English | MEDLINE | ID: mdl-22384042

ABSTRACT

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.


Subject(s)
Aromatic Amino Acid Decarboxylase Inhibitors , Parkinson Disease/drug therapy , Animals , Catalytic Domain , Chemistry, Pharmaceutical/methods , Databases, Factual , Dopamine/metabolism , Dose-Response Relationship, Drug , Drug Design , Humans , In Vitro Techniques , Inhibitory Concentration 50 , Kinetics , Models, Chemical , Models, Molecular , Molecular Conformation , Protein Binding , Serotonin/metabolism , Swine
16.
Int J Biochem Cell Biol ; 44(3): 536-46, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22198249

ABSTRACT

Alanine:glyoxylate aminotransferase (AGT) is a pyridoxal-phosphate (PLP)-dependent enzyme. Its deficiency causes the hereditary kidney stone disease primary hyperoxaluria type 1. AGT is a highly stable compact dimer and the first 21 residues of each subunit form an extension which wraps over the surface of the neighboring subunit. Naturally occurring and artificial amino acid replacements in this extension create changes in the functional properties of AGT in mammalian cells, including relocation of the enzyme from peroxisomes to mitochondria. In order to elucidate the structural and functional role of this N-terminal extension, we have analyzed the consequences of its removal using a variety of biochemical and cell biological methods. When expressed in Escherichia coli, the N-terminal deleted form of AGT showed the presence of the protein but in an insoluble form resulting in only a 10% soluble yield as compared to the full-length version. The purified soluble fraction showed reduced affinity for PLP and greatly reduced catalytic activity. Although maintaining a dimer form, it was highly prone to self-aggregation. When expressed in a mammalian cell line, the truncated construct was normally targeted to peroxisomes, where it formed large stable but catalytically inactive aggregates. These results suggest that the N-terminal extension plays an essential role in allowing AGT to attain its correct conformation and functional activity. The precise mechanism of this effect is still under investigation.


Subject(s)
Hyperoxaluria, Primary/genetics , Liver/enzymology , Transaminases/chemistry , Animals , CHO Cells , Cloning, Molecular , Cricetinae , Humans , Mice , Peptide Fragments/genetics , Protein Conformation , Protein Folding , Protein Multimerization , Sequence Deletion/genetics , Transaminases/genetics
17.
Mol Genet Metab ; 105(1): 132-40, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22018727

ABSTRACT

Primary Hyperoxaluria Type I (PH1) is a disorder of glyoxylate metabolism caused by mutations in the human AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) dependent enzyme. Previous investigations highlighted that, although PH1 is characterized by a significant variability in terms of enzymatic phenotype, the majority of the pathogenic variants are believed to share both structural and functional defects, as mainly revealed by data on AGT activity and expression level in crude cellular extracts. However, the knowledge of the defects of the AGT variants at a protein level is still poor. We therefore performed a side-by-side comparison between normal AGT and nine purified recombinant pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization, and thermal stability of both the holo- and apo-forms. Notably, we chose four variants in which the mutated residues are located in the large domain of AGT either within the active site and interacting with the coenzyme or in its proximity, and five variants in which the mutated residues are distant from the active site either in the large or in the small domain. Overall, this integrated analysis of enzymatic activity, spectroscopic and stability information is used to (i) reassess previous data obtained with crude cellular extracts, (ii) establish which form(s) (i.e. holoenzyme and/or apoenzyme) and region(s) (i.e. active site microenvironment, large and/or small domain) of the protein are affected by each mutation, and (iii) suggest the possible therapeutic approach for patients bearing the examined mutations.


Subject(s)
Apoproteins/genetics , Hyperoxaluria, Primary/enzymology , Hyperoxaluria, Primary/genetics , Mutant Proteins/genetics , Mutation/genetics , Transaminases/genetics , Animals , Apoproteins/chemistry , Circular Dichroism , Computational Biology , Enzyme Stability , Escherichia coli , Humans , Kinetics , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Pyridoxal Phosphate/metabolism , Rabbits , Temperature , Transaminases/chemistry , Transaminases/isolation & purification
18.
J Inherit Metab Dis ; 34(6): 1213-24, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21541720

ABSTRACT

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.


Subject(s)
Amino Acid Metabolism, Inborn Errors/genetics , Decarboxylation/physiology , Dopa Decarboxylase/genetics , Models, Molecular , Mutagenesis, Site-Directed , Point Mutation , Amino Acid Metabolism, Inborn Errors/enzymology , Amino Acid Metabolism, Inborn Errors/pathology , Aromatic-L-Amino-Acid Decarboxylases/deficiency , Aromatic-L-Amino-Acid Decarboxylases/genetics , Dopa Decarboxylase/chemistry , Dopa Decarboxylase/isolation & purification , Humans , Kinetics , Molecular Structure , Mutagenesis, Site-Directed/methods , Point Mutation/genetics , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
19.
Biochemistry ; 47(27): 7187-95, 2008 Jul 08.
Article in English | MEDLINE | ID: mdl-18547057

ABSTRACT

The unusual oxygen-consuming oxidative deamination reaction catalyzed by the pyridoxal 5'-phosphate (PLP) enzyme DOPA decarboxylase (DDC) was here investigated. Either wild-type or Y332F DDC variant is able to perform such oxidation toward aromatic amines or aromatic l-amino acids, respectively, without the aid of any cofactor related to oxygen chemistry. Oxidative deamination produces, in equivalent amounts, a carbonyl compound and ammonia, accompanied by dioxygen consumption in a 1:2 molar ratio with respect to the products. Kinetic studies either in the pre-steady or in the steady state, together with HPLC analyses of reaction mixtures under varying experimental conditions, revealed that a ketimine accumulates during the linear phase of product formation. This species is reactive since it is converted back to PLP when the substrate is consumed. Rapid-mixing chemical quench studies provide evidence that the ketimine is indeed an intermediate formed during the first catalytic cycle. Moreover, superoxide anion and hydrogen peroxide are both generated during the catalytic cycles. On this basis, a mechanism of oxidative deamination consistent with the present data is proposed. Furthermore, the catalytic properties of the T246A DDC mutant together with those previously obtained with H192Q mutant allow us to propose that the Thr246-His192 dyad could act as a general base in promoting the first step of the oxidative deamination of aromatic amines.


Subject(s)
Dopa Decarboxylase/metabolism , Aerobiosis , Amino Acids/metabolism , Anaerobiosis , Animals , Binding Sites , Catalysis , Cattle , Deamination , Dihydroxyphenylalanine/chemistry , Dihydroxyphenylalanine/metabolism , Horses , Mutant Proteins/metabolism , Oxidation-Reduction , Oxygen/metabolism , Oxygen Consumption , Pyridoxamine/analogs & derivatives , Pyridoxamine/metabolism , Substrate Specificity , Swine , Time Factors
20.
Biochim Biophys Acta ; 1784(9): 1356-62, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18492492

ABSTRACT

In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-L-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with L-alanine. On the other hand, during the reaction of AGT with L-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of L-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of L-cysteine is proposed. L-cysteine partitions between these two reactions with a ratio of approximately 2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of L-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of L-alanine and L-serine, respectively. Conversely, L-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of L-alanine and L-serine, respectively. This appears to be consistent with the calculated interaction energies of the L-cysteine, L-alanine and L-serine docked at the active site of AGT.


Subject(s)
Transaminases/chemistry , Transaminases/metabolism , Catalytic Domain , Cysteine/metabolism , Humans , In Vitro Techniques , Kinetics , Liver/enzymology , Models, Biological , Models, Molecular , Peroxisomes/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrophotometry , Substrate Specificity , Transaminases/genetics , beta-Alanine/analogs & derivatives , beta-Alanine/metabolism
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