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

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

Phosphorylation of pyridoxal 5'-phosphate enzymes: an intriguing and neglected topic.

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.

The novel R347g pathogenic mutation of aromatic amino acid decarboxylase provides additional molecular insights into enzyme catalysis and deficiency.

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.

Use of polymer conjugates for the intraperoxisomal delivery of engineered human alanine:glyoxylate aminotransferase as a protein therapy for primary hyperoxaluria type I.

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficit causes the rare disorder Primary Hyperoxaluria Type I (PH1). We now describe the conjugation of poly(ethylene glycol)-co-poly(L-glutamic acid) (PEG-PGA) block-co-polymer to AGT via the formation of disulfide bonds between the polymer and solvent-exposed cysteine residues of the enzyme. PEG-PGA conjugation did not affect AGT structural/functional properties and allowed the enzyme to be internalized in a cellular model of PH1 and to restore glyoxylate-detoxification. The insertion of the C387S/K390S amino acid substitutions, known to favor interaction with the peroxisomal import machinery, reduced conjugation efficiency, but endowed conjugates with the ability to reach the peroxisomal compartment. These results, along with the finding that conjugates are hemocompatible, stable in plasma, and non-immunogenic, hold promise for the development of polypeptide-based AGT conjugates as a therapeutic option for PH1 patients and represent the base for applications to other diseases related to deficits in peroxisomal proteins.

A comprehensive picture of the mutations associated with aromatic amino acid decarboxylase deficiency: from molecular mechanisms to therapy implications.

Dopa decarboxylase (DDC), or aromatic amino acid decarboxylase (AADC), is a pyridoxal 5'-phosphate enzyme responsible for the production of the neurotransmitters dopamine and serotonin. Deficit of this enzyme causes AADC deficiency, an inherited neurometabolic disorder. To date, 18 missense homozygous mutations have been identified through genetic screening in ∼80 patients. However, little is known about the mechanism(s) by which mutations cause disease. Here we investigated the impact of these pathogenic mutations and of an artificial one on the conformation and the activity of wild-type DDC by a combined approach of bioinformatic, spectroscopic and kinetic analyses. All mutations reduce the kcat value, and, except the mutation R347Q, alter the tertiary structure, as revealed by an increased hydrophobic surface and a decreased near-UV circular dichroism signal. The integrated analysis of the structural and functional consequences of each mutation strongly suggests that the reason underlying the pathogenicity of the majority of disease-causing mutations is the incorrect apo-holo conversion. In fact, the most remarkable effects are seen upon mutation of residues His70, His72, Tyr79, Phe80, Pro81, Arg462 and Arg447 mapping to or directly interacting with loop1, a structural key element involved in the apo-holo switch. Instead, different mechanisms are responsible for the pathogenicity of R347Q, a mere catalytic mutation, and of L38P and A110Q mutations causing structural-functional defects. These are due to local perturbation transmitted to the active site, as predicted by molecular dynamic analyses. Overall, the results not only give comprehensive molecular insights into AADC deficiency, but also provide an experimental framework to suggest appropriate therapeutic treatments.

S250F variant associated with aromatic amino acid decarboxylase deficiency: molecular defects and intracellular rescue by pyridoxine.

Dopa or aromatic amino acid decarboxylase (DDC, AADC) is a pyridoxal 5'-phosphate-dependent enzyme that catalyses the production of the neurotransmitters dopamine and serotonin. Among the so far identified mutations associated with AADC deficiency, an inherited rare neurometabolic disease, the S250F mutation is the most frequent one. Here, for the first time, the molecular basis of the deficit of the S250F variant was investigated both in vitro and in cellular systems. Ser250 is not essential for the catalytic activity of the enzyme. However, its mutation to Phe causes a ~7-fold reduction of catalytic efficiency and a conformational change in the proximity of the mutated residue that is transmitted to the active site. In cellular extracts of E. coli and mammalian cells, both the specific activity and the protein level of the variant decrease with respect to the wild-type. The results with mammalian cells indicate that the mutation does not affect intracellular mRNA levels, and are consistent with a model where S250F undergoes a degradation process via the proteasome, possibly through an ubiquitination process occurring faster than in the wild-type. Overall, biochemical and cell biology experiments show that loss of function of S250F occurs by two distinct but not exclusive mechanisms affecting activity and folding. Importantly, 4-phenylbutirric acid (4-PBA) or, to a major extent, pyridoxine increase the expression level and, in a dose-dependent manner, the decarboxylase specific activity of mutant-expressing cells. This strongly suggests that 4-PBA and/or pyridoxine administration may be of important value in therapy of patients bearing the S250F mutation.

Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to Group II decarboxylases.

DOPA decarboxylase, the dimeric enzyme responsible for the synthesis of neurotransmitters dopamine and serotonin, is involved in severe neurological diseases such as Parkinson disease, schizophrenia, and depression. Binding of the pyridoxal-5'-phosphate (PLP) cofactor to the apoenzyme is thought to represent a central mechanism for the regulation of its activity. We solved the structure of the human apoenzyme and found it exists in an unexpected open conformation: compared to the pig kidney holoenzyme, the dimer subunits move 20 Å apart and the two active sites become solvent exposed. Moreover, by tuning the PLP concentration in the crystals, we obtained two more structures with different conformations of the active site. Analysis of three-dimensional data coupled to a kinetic study allows to identify the structural determinants of the open/close conformational change occurring upon PLP binding and thereby propose a model for the preferential degradation of the apoenzymes of Group II decarboxylases.

Crystal structure of the S187F variant of human liver alanine: glyoxylate [corrected] aminotransferase associated with primary hyperoxaluria type I and its functional implications.

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5'-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT-pyridoxamine 5'-phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300- to 500-fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results.

Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I.

G41 is an interfacial residue located within the alpha-helix 34-42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (approximately 5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 --> R mutation causes a partial unwinding of the 34-42 alpha-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.

Human liver peroxisomal alanine:glyoxylate aminotransferase: characterization of the two allelic forms and their pathogenic variants.

The hepatic peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-enzyme whose deficiency is responsible for Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive disorder. In the last few years the knowledge of the characteristics of AGT and the transfer of this information into some pathogenic variants have significantly contributed to the improvement of the understanding at the molecular level of the PH1 pathogenesis. In this review, the spectroscopic features, the coenzyme's binding affinity, the steady-state kinetic parameters as well as the sensitivity to thermal and chemical stress of the two allelic forms of AGT, the major (AGT-Ma) and the minor (AGT-Mi) allele, have been described. Moreover, we summarize the characterization obtained by means of biochemical and bioinformatic analyses of the following PH1-causing variants in the recombinant purified forms: G82E associated with the major allele, F152I encoded on the background of the minor allele, and the G41 mutants which co-segregate either with the major allele (G41R-Ma and G41V-Ma) or with the minor allele (G41R-Mi). The data have been correlated with previous clinical and cell biology results, which allow us to (i) highlight the functional differences between AGT-Ma and AGT-Mi, (ii) identify the structural and functional molecular defects of the pathogenic variants, (iii) improve the correlation between the genotype and the enzymatic phenotype, (iv) foresee or understand the molecular basis of the responsiveness to pyridoxine treatment of patients bearing these mutations, and (v) pave the way for new treatment strategies. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

Molecular and Cellular Studies Reveal Folding Defects of Human Ornithine Aminotransferase Variants Associated With Gyrate Atrophy of the Choroid and Retina.

The deficit of human ornithine aminotransferase (hOAT) is responsible for gyrate atrophy (GA), a rare recessive inherited disorder. Although more than 60 disease-associated mutations have been identified to date, the molecular mechanisms explaining how each mutation leads to the deficit of OAT are mostly unknown. To fill this gap, we considered six representative missense mutations present in homozygous patients concerning residues spread over the hOAT structure. E. coli expression, spectroscopic, kinetic and bioinformatic analyses, reveal that the R154L and G237D mutations induce a catalytic more than a folding defect, the Q90E and R271K mutations mainly impact folding efficiency, while the E318K and C394Y mutations give rise to both folding and catalytic defects. In a human cellular model of disease folding-defective variants, although at a different extent, display reduced protein levels and/or specific activity, due to increased aggregation and/or degradation propensity. The supplementation with Vitamin B6, to mimic a treatment strategy available for GA patients, does not significantly improve the expression/activity of folding-defective variants, in contrast with the clinical responsiveness of patients bearing the E318K mutation. Thus, we speculate that the action of vitamin B6 could be also independent of hOAT. Overall, these data represent a further effort toward a comprehensive analysis of GA pathogenesis at molecular and cellular level, with important relapses for the improvement of genotype/phenotype correlations and the development of novel treatments.

An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme.

Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 microM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed.

Multiple roles of the active site lysine of Dopa decarboxylase.

The pyridoxal 5'-phosphate dependent-enzyme Dopa decarboxylase, responsible for the irreversible conversion of l-Dopa to dopamine, is an attractive drug target. The contribution of the pyridoxal-Lys303 to the catalytic mechanisms of decarboxylation and oxidative deamination is analyzed. The K303A variant binds the coenzyme with a 100-fold decreased apparent equilibrium binding affinity with respect to the wild-type enzyme. Unlike the wild-type, K303A in the presence of l-Dopa displays a parallel progress course of formation of both dopamine and 3,4-dihydroxyphenylacetaldehyde (plus ammonia) with a burst followed by a linear phase. Moreover, the finding that the catalytic efficiencies of decarboxylation and of oxidative deamination display a decrease of 1500- and 17-fold, respectively, with respect to the wild-type, is indicative of a different impact of Lys303 mutation on these reactions. Kinetic analyses reveal that Lys303 is involved in external aldimine formation and hydrolysis as well as in product release which affects the rate-determining step of decarboxylation.

Construction, purification and characterization of untagged human liver alanine-glyoxylate aminotransferase expressed in Escherichia coli.

His-tagging is commonly used to aid and expedite the purification of recombinant proteins. It is commonly assumed, though less frequently tested, that the His-tag affects neither the structure nor the stability of the protein. Alanine:glyoxylate aminotransferase (AGT) is a peroxisomal pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the transamination of alanine and glyoxylate to pyruvate and glycine. AGT is a clinically relevant enzyme whose deficiency causes an inherited rare metabolic disorder named primary hyperoxaluria type I. Until now, the structure and function of this enzyme have been studied using recombinant wild-type AGT and variants purified using a hexa-histidine tag. However, the study of the functional roles of the N- and C-termini in the dimerization process and on the import into the peroxisome, respectively, requires the preparation of human liver AGT without histidine tags. We report for the first time the expression of untagged AGT together with a new rapid protocol for its purification. In addition, the kinetic parameters for the forward and reverse transamination catalyzed by untagged AGT as well as the spectroscopic features, the K(D(PLP)), the pH and thermal stability of the enzyme in the holo- and apo-form have been determined. This investigation will be the starting point for a detailed understanding of the contributions of the N- and C-termini on the dimerization and folding of AGT, and on its import into the peroxisome. This is prerequisite to understand how pathological mutations affect the proper native quaternary and tertiary structure, stability, and targeting of the enzyme.

Mapping of human autoantibody epitopes on aromatic L-amino acid decarboxylase.

CONTEXT: Aromatic l-amino acid decarboxylase (AADC) is target of autoantibodies in autoimmune polyendocrine syndrome I (APS I), especially in patients with autoimmune hepatitis. Little information is currently available on AADC autoantibody epitopes and on the interrelation between autoantibody-mediated inhibition of enzymatic activity and epitope specificity. DESIGN: We tested the immunoreactivity of full-length porcine AADC and of eight fragments of the enzyme with human serum from 18 patients with APS I, 199 with non-APS I autoimmune Addison's disease, 124 with type 1 diabetes mellitus, 36 with Graves' disease, and 141 healthy control subjects, and we evaluated the autoantibody-mediated enzymatic inhibition. RESULTS: AADC antibodies (Ab) were detected in 12 of 18 (67%) APS I patients and in six of 199 (3%) autoimmune Addison's disease patients. Four patients with autoimmune hepatitis were all positive for AADCAb. None of the 141 healthy control subjects, 82 patients with nonautoimmune adrenal insufficiency, 124 with type 1 diabetes mellitus, and 36 with Graves' disease were found positive. Two epitope regions, corresponding to amino acids 274-299 (E1) and 380-471 (E2) were identified. Localization of E1 was confirmed by displacement studies with synthetic peptides corresponding to peptides of porcine AADC. All 12 AADCAb-positive APS I sera reacted with E1, and seven of 12 (58%) reacted also with E2. E2-specific, but not E1-specific, autoantibodies were associated with a significant inhibition of in vitro AADC enzymatic activity. CONCLUSIONS: We mapped the human AADCAb epitopes to the middle and COOH-terminal regions of the enzyme. Autoantibodies to the COOH-terminal region induce a significant inhibition of enzymatic activity.

New Insights Emerging from Recent Investigations on Human Group II Pyridoxal 5'-Phosphate Decarboxylases.

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).

Radiation damage at the active site of human alanine:glyoxylate aminotransferase reveals that the cofactor position is finely tuned during catalysis.

The alanine:glyoxylate aminotransferase (AGT), a hepatocyte-specific pyridoxal-5'-phosphate (PLP) dependent enzyme, transaminates L-alanine and glyoxylate to glycine and pyruvate, thus detoxifying glyoxylate and preventing pathological oxalate precipitation in tissues. In the widely accepted catalytic mechanism of the aminotransferase family, the lysine binding to PLP acts as a catalyst in the stepwise 1,3-proton transfer, interconverting the external aldimine to ketimine. This step requires protonation by a conserved aspartate of the pyridine nitrogen of PLP to enhance its ability to stabilize the carbanionic intermediate. The aspartate residue is also responsible for a significant geometrical distortion of the internal aldimine, crucial for catalysis. We present the structure of human AGT in which complete X-ray photoreduction of the Schiff base has occurred. This result, together with two crystal structures of the conserved aspartate pathogenic variant (D183N) and the molecular modeling of the transaldimination step, led us to propose that an interplay of opposite forces, which we named spring mechanism, finely tunes PLP geometry during catalysis and is essential to move the external aldimine in the correct position in order for the 1,3-proton transfer to occur.

A quinonoid is an intermediate of oxidative deamination reaction catalyzed by Dopa decarboxylase.

The reactions of Dopa decarboxylase (DDC) with l- and d-enantiomers of tryptophan methyl ester are described. Although both the enantiomers bind to the active site of the enzyme with similar affinity, their binding modes are different. l-enantiomer binds in an unproductive mode, while d-enantiomer acts as an oxidative deamination substrate. For the first time a quinonoid has been detected as intermediate of this reaction. By using rapid-scanning stopped-flow kinetic technique rate constants for formation and decay of this species have been determined. All these data, besides validating the functional DDC active site model, represent an important step toward the elucidation of the catalytic pathway of oxidative deamination.

The Chaperoning Activity of Amino-oxyacetic Acid on Folding-Defective Variants of Human Alanine:Glyoxylate Aminotransferase Causing Primary Hyperoxaluria Type I.

The rare disease Primary Hyperoxaluria Type I (PH1) results from the deficit of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene frequently leading to protein misfolding. Pharmacological chaperone (PC) therapy is a newly developed approach for misfolding diseases based on the use of small molecule ligands able to promote the correct folding of a mutant enzyme. In this report, we describe the interaction of amino-oxyacetic acid (AOA) with the recombinant purified form of two polymorphic species of AGT, AGT-Ma and AGT-Mi, and with three pathogenic variants bearing previously identified folding defects: G41R-Ma, G170R-Mi, and I244T-Mi. We found that for all these enzyme AOA (i) forms an oxime at the active site, (ii) behaves as a slow, tight-binding inhibitor with KI values in the nanomolar range, and (iii) increases the thermal stability. Furthermore, experiments performed in mammalian cells revealed that AOA acts as a PC by partly preventing the intracellular aggregation of G41R-Ma and by promoting the correct peroxisomal import of G170R-Mi and I244T-Mi. Based on these data, we carried out a small-scale screening campaign. We identified four AOA analogues acting as AGT inhibitors, even if only one was found to act as a PC. The possible relationship between the structure and the PC activity of these compounds is discussed. Altogether, these results provide the proof-of-principle for the feasibility of a therapy with PCs for PH1-causing variants bearing folding defects and provide the scaffold for the identification of more specific ligands.

The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed.