Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bou... more Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bound at the active site with great affinity. From a comparison of the interaction of a number of such compounds with the apoenzyme the delta G0 values for the binding of the substrate carboxy and phenyl groups and of the coenzyme aldehydic group were determined to be equal to (or more negative than) ‒3.8. ‒8.4 and ‒12.5kJ/mol (-0.9, ‒1.9 and ‒3kcal/mol) respectively; the delta G0 for the binding of the coenzyme phosphate group was shown to be more negative than ‒20.5kJ/mol (-4.9kcal/mol). Two features of the binding process of the coenzyme-substrate analogues to tyrosine decarboxylase have already been found in the case of tyrosine aminotransferase [Borri-Voltattorni, Orlacchio, Giartosio, Conti & Turano (1975) Eur. J. Biochem. 53, 151-160]: (1) in the binding of the substrate to the enzyme a significant fraction of the instrinsic delta G0 appears to be used for some associated endoergonic...
The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichr... more The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichroism, and fluorescence of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase from pig kidney (pkDDC) was studied under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M GuCl, gives rise to a dimeric inactive species which has lost pyridoxal 5′-phosphate (PLP), and has a high tendency to aggregate, but retains almost all of the native spectroscopic characteristics. The second equilibrium transition, between 1 and 2.2 M GuCl, involves dimer dissociation, with some loss of tertiary and secondary structure. Additionally, gross conformational changes at or near the PLP microenvironment were detected by fluorescence of NaBH4-reduced enzyme. The third step, presumably representing complete unfolding of pkDDC, appears to be complete at 4.5 M GuCl, as indicated by the lack of further substantial changes in any of the signals being ...
DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine a... more DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinson's disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.
The present study has explained the general reaction mechanism of the bacterial tyrosine decarbox... more The present study has explained the general reaction mechanism of the bacterial tyrosine decarboxylase. The rate equation for this mechanism has been presented. The steady state kinetics of tyrosine decarboxylase, as for tyrosine transaminase, have shown that the apoenzyme can bind not only the coenzyme, but also the non-enzymatically formed Schiff base between the coenzyme and the substrate. Our data then have confirmed the importance of the non-enzymatically formed Schiff base in the B6-dependent enzymes, possibly in all of them which have a low affinity constant for the coenzyme, such that the coenzyme must be present in excess in respect to the protein to saturate the active center. The interaction between apotyrosine decarboxylase with pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate has been studied.
Primary hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:gl... more 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, while 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.
Two fragments (Mr =38,000 and 14,000)derived from limited trypsin treatment of pig kidney Dopa de... more Two fragments (Mr =38,000 and 14,000)derived from limited trypsin treatment of pig kidney Dopa decarboxylase have been separated in denaturing conditions. The PLP-binding site has been located on the larger fragment. Results of sequence analysis by automated Edman degradation of the intact enzyme compared with those of the two fragments indicate that the cleavage takes place at about one-third from the COOH-terminal. A 50 residue NH2-terminal of the smaller fragment has been determined.
A procedure for 3,4-dihydroxyphenylalanine decarboxylase from pig kidney purification is describe... more A procedure for 3,4-dihydroxyphenylalanine decarboxylase from pig kidney purification is described in detail. The preparation has no detectable impurity on electrophoresis and on ultracentrifugation and has a coenzyme content and a specific activity comparable with the same enzyme purified by other authors. However two significant differences are observed: a different stimulation of activity by added pyridoxal 5'-phosphate and a nearly complete decarboxylation of ~-3,4-dihydroxyphenylalanine in absence of added coenzyme. Absorption, fluorescence and circular dichroism properties of the coenzyme-apoenzyme interaction are also described. The results are consistent with the existence of at least four coenzyme-apoenzyme complexes, three of them active. 3,4-Dihydroxyphenylalanine decarboxylase, also called aromatic amino acid decarboxylase owing to its poor substrate specificity [I] has been purified from pig kidney by at least three different methods [I-31. Some properties of the three preparations like molecular weight, subunit composition [l, 21 and specific activity [I-31 are significantly different. Recently some kinetic data have been presented [4] which suggest a mechanism for the regulation of 3,4dihydroxyphenylalanine decarboxylase. Also these kinetic data cannot be reproduced by using a different method [3] of enzyme purification, as it will be shown in this paper. It seems that some structural and functional features of 3,4-dihydroxyphenylalanine decarboxylase are critically dependent on some steps of the purification procedure. It is worthwhile therefore to provide a detailed description of the procedure used by us [3], never given before. Some properties of the enzyme obtained in this way will also be described, with a particular emphasis on the coenzyme-apoenzyme interaction. MATERIALS AND METHODS ~-3,4-Dihydroxyphenylalanine, pyridoxal-P, dithiothreitol, 2,4,6-trinitrobenzene-l-sulphonic acid Abbreviations. Pyridoxal-P, pyridoxal-5'-phosphate; QAE-Se-Enzyme. 3,4-Dihydroxyphenylalanine decarboxylase 01' aro-phadex, quaternary aminoethyl-Sephadex. matic L-amino-acid decarboxylase (EC 4.1 J.28).
Residues D271, H192, H302 and N300 of L-3,4-dihydroxyphenylalanine decarboxylase (DDC), a homodim... more Residues D271, H192, H302 and N300 of L-3,4-dihydroxyphenylalanine decarboxylase (DDC), a homodimeric pyridoxal 5'-phosphate (PLP) enzyme, were mutated in order to acquire information on the catalytic mechanism. These residues are potential participants in catalysis because they belong to the common PLP-binding structural motif of group I, II and III decarboxylases and other PLP enzymes, and because they are among the putative active-site residues of structural modelled rat liver DDC. The spectroscopic features of the D271E, H192Q, H302Q and N300A mutants as well as their dissociation constants for PLP suggest that substitution of each of these residues causes alteration of the state of the bound coenzyme molecule and of the conformation of aromatic amino acids, possibly in the vicinity of the active site. This supports, but does not prove, the possibility that these residues are located in the coenzyme-binding cleft. Interestingly, mutation of each residue generates an oxidative decarboxylase activity towards L-3,4-dihydroxyphenylalanine (L-Dopa), not inherent in the wild-type in aerobiosis, and reduces the nonoxidative decarboxylase activity of L-Dopa from 3- to 390-fold. The partition ratio between oxidative and nonoxidative decarboxylation ranges from 5.7 x 10(-4) for N300A mutant to 946 x 10(-4) for H302Q mutant. Unlike wild-type enzyme, the mutants catalyse these two reactions to the same extent either in the presence or absence of O2. In addition, all four mutants exhibit an extremely low level of the oxidative deaminase activity towards serotonin with respect to wild-type. All these findings demonstrate that although D271, H192, H302 and N300 are not essential for catalysis, mutation of these residues alters the nature of catalysis. A possible relationship among the integrity of the PLP cleft, the productive binding of O2 and the transition to a closed conformational state of DDC is discussed.
Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but als... more Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O 2 consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O 2 bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O 2. The results are consistent with a mechanism in which 5-HT binds to DDC before O 2. The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of ∼7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K) 5-HT profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the ␣-amino group of 5-HT. No titrable ionizing residue was detected in the (V/K) O 2 profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5Ј-phosphate formation increases below a single pK of ∼6.7. This value mirrors the spectrophotometric pK spec of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.
Dopa decarboxylase (DDC), a pyridoxal 59-phosphate (PLP) enzyme responsible for the biosynthesis ... more Dopa decarboxylase (DDC), a pyridoxal 59-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 virtualscreening (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 mM. 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.
The Journal of Clinical Endocrinology & Metabolism, 2007
Aromatic l-amino acid decarboxylase (AADC) is target of autoantibodies in autoimmune polyendocrin... more 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. 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. 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. 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.
Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5-phosphate (PLP... more Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe 152 variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe 152 variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400fold lower. Thus, Phe 152 is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp 108 , as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.
The vitamin B 6-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a larg... more The vitamin B 6-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in amino acid metabolism. These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and protein structural organization. Almost 1.5% of all genes in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of enzyme-catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), serine hydroxymethyltransferase (tumors and malaria), ornithine decarboxylase (African sleeping sickness and, potentially, tumors), alanine racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or metabolic pathway, the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and ongoing research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.
The effect of N-(5'-phosphopyridoxyl)-L-5-hydroxytryptophan, N-(5'-phosphopyridox... more The effect of N-(5'-phosphopyridoxyl)-L-5-hydroxytryptophan, N-(5'-phosphopyridoxyl)-D-5-hydroxytryptophan and N-(5'-phosphopyridoxyl)-5-hydroxytryptamine on the reactivation of apoDopa decarboxylase to holoenzyme has been investigated. The different degree of inhibition exerted by these adducts has been interpreted on the basis of a different orientation of the 2 isomers of 5-HTP at the active of Dopa decarboxylase.
The unusual oxygen-consuming oxidative deamination reaction catalyzed by the pyridoxal 5′-phospha... more 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.
Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bou... more Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bound at the active site with great affinity. From a comparison of the interaction of a number of such compounds with the apoenzyme the delta G0 values for the binding of the substrate carboxy and phenyl groups and of the coenzyme aldehydic group were determined to be equal to (or more negative than) ‒3.8. ‒8.4 and ‒12.5kJ/mol (-0.9, ‒1.9 and ‒3kcal/mol) respectively; the delta G0 for the binding of the coenzyme phosphate group was shown to be more negative than ‒20.5kJ/mol (-4.9kcal/mol). Two features of the binding process of the coenzyme-substrate analogues to tyrosine decarboxylase have already been found in the case of tyrosine aminotransferase [Borri-Voltattorni, Orlacchio, Giartosio, Conti & Turano (1975) Eur. J. Biochem. 53, 151-160]: (1) in the binding of the substrate to the enzyme a significant fraction of the instrinsic delta G0 appears to be used for some associated endoergonic...
The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichr... more The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichroism, and fluorescence of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase from pig kidney (pkDDC) was studied under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M GuCl, gives rise to a dimeric inactive species which has lost pyridoxal 5′-phosphate (PLP), and has a high tendency to aggregate, but retains almost all of the native spectroscopic characteristics. The second equilibrium transition, between 1 and 2.2 M GuCl, involves dimer dissociation, with some loss of tertiary and secondary structure. Additionally, gross conformational changes at or near the PLP microenvironment were detected by fluorescence of NaBH4-reduced enzyme. The third step, presumably representing complete unfolding of pkDDC, appears to be complete at 4.5 M GuCl, as indicated by the lack of further substantial changes in any of the signals being ...
DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine a... more DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinson's disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.
The present study has explained the general reaction mechanism of the bacterial tyrosine decarbox... more The present study has explained the general reaction mechanism of the bacterial tyrosine decarboxylase. The rate equation for this mechanism has been presented. The steady state kinetics of tyrosine decarboxylase, as for tyrosine transaminase, have shown that the apoenzyme can bind not only the coenzyme, but also the non-enzymatically formed Schiff base between the coenzyme and the substrate. Our data then have confirmed the importance of the non-enzymatically formed Schiff base in the B6-dependent enzymes, possibly in all of them which have a low affinity constant for the coenzyme, such that the coenzyme must be present in excess in respect to the protein to saturate the active center. The interaction between apotyrosine decarboxylase with pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate has been studied.
Primary hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:gl... more 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, while 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.
Two fragments (Mr =38,000 and 14,000)derived from limited trypsin treatment of pig kidney Dopa de... more Two fragments (Mr =38,000 and 14,000)derived from limited trypsin treatment of pig kidney Dopa decarboxylase have been separated in denaturing conditions. The PLP-binding site has been located on the larger fragment. Results of sequence analysis by automated Edman degradation of the intact enzyme compared with those of the two fragments indicate that the cleavage takes place at about one-third from the COOH-terminal. A 50 residue NH2-terminal of the smaller fragment has been determined.
A procedure for 3,4-dihydroxyphenylalanine decarboxylase from pig kidney purification is describe... more A procedure for 3,4-dihydroxyphenylalanine decarboxylase from pig kidney purification is described in detail. The preparation has no detectable impurity on electrophoresis and on ultracentrifugation and has a coenzyme content and a specific activity comparable with the same enzyme purified by other authors. However two significant differences are observed: a different stimulation of activity by added pyridoxal 5'-phosphate and a nearly complete decarboxylation of ~-3,4-dihydroxyphenylalanine in absence of added coenzyme. Absorption, fluorescence and circular dichroism properties of the coenzyme-apoenzyme interaction are also described. The results are consistent with the existence of at least four coenzyme-apoenzyme complexes, three of them active. 3,4-Dihydroxyphenylalanine decarboxylase, also called aromatic amino acid decarboxylase owing to its poor substrate specificity [I] has been purified from pig kidney by at least three different methods [I-31. Some properties of the three preparations like molecular weight, subunit composition [l, 21 and specific activity [I-31 are significantly different. Recently some kinetic data have been presented [4] which suggest a mechanism for the regulation of 3,4dihydroxyphenylalanine decarboxylase. Also these kinetic data cannot be reproduced by using a different method [3] of enzyme purification, as it will be shown in this paper. It seems that some structural and functional features of 3,4-dihydroxyphenylalanine decarboxylase are critically dependent on some steps of the purification procedure. It is worthwhile therefore to provide a detailed description of the procedure used by us [3], never given before. Some properties of the enzyme obtained in this way will also be described, with a particular emphasis on the coenzyme-apoenzyme interaction. MATERIALS AND METHODS ~-3,4-Dihydroxyphenylalanine, pyridoxal-P, dithiothreitol, 2,4,6-trinitrobenzene-l-sulphonic acid Abbreviations. Pyridoxal-P, pyridoxal-5'-phosphate; QAE-Se-Enzyme. 3,4-Dihydroxyphenylalanine decarboxylase 01' aro-phadex, quaternary aminoethyl-Sephadex. matic L-amino-acid decarboxylase (EC 4.1 J.28).
Residues D271, H192, H302 and N300 of L-3,4-dihydroxyphenylalanine decarboxylase (DDC), a homodim... more Residues D271, H192, H302 and N300 of L-3,4-dihydroxyphenylalanine decarboxylase (DDC), a homodimeric pyridoxal 5'-phosphate (PLP) enzyme, were mutated in order to acquire information on the catalytic mechanism. These residues are potential participants in catalysis because they belong to the common PLP-binding structural motif of group I, II and III decarboxylases and other PLP enzymes, and because they are among the putative active-site residues of structural modelled rat liver DDC. The spectroscopic features of the D271E, H192Q, H302Q and N300A mutants as well as their dissociation constants for PLP suggest that substitution of each of these residues causes alteration of the state of the bound coenzyme molecule and of the conformation of aromatic amino acids, possibly in the vicinity of the active site. This supports, but does not prove, the possibility that these residues are located in the coenzyme-binding cleft. Interestingly, mutation of each residue generates an oxidative decarboxylase activity towards L-3,4-dihydroxyphenylalanine (L-Dopa), not inherent in the wild-type in aerobiosis, and reduces the nonoxidative decarboxylase activity of L-Dopa from 3- to 390-fold. The partition ratio between oxidative and nonoxidative decarboxylation ranges from 5.7 x 10(-4) for N300A mutant to 946 x 10(-4) for H302Q mutant. Unlike wild-type enzyme, the mutants catalyse these two reactions to the same extent either in the presence or absence of O2. In addition, all four mutants exhibit an extremely low level of the oxidative deaminase activity towards serotonin with respect to wild-type. All these findings demonstrate that although D271, H192, H302 and N300 are not essential for catalysis, mutation of these residues alters the nature of catalysis. A possible relationship among the integrity of the PLP cleft, the productive binding of O2 and the transition to a closed conformational state of DDC is discussed.
Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but als... more Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O 2 consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O 2 bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O 2. The results are consistent with a mechanism in which 5-HT binds to DDC before O 2. The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of ∼7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K) 5-HT profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the ␣-amino group of 5-HT. No titrable ionizing residue was detected in the (V/K) O 2 profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5Ј-phosphate formation increases below a single pK of ∼6.7. This value mirrors the spectrophotometric pK spec of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.
Dopa decarboxylase (DDC), a pyridoxal 59-phosphate (PLP) enzyme responsible for the biosynthesis ... more Dopa decarboxylase (DDC), a pyridoxal 59-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 virtualscreening (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 mM. 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.
The Journal of Clinical Endocrinology & Metabolism, 2007
Aromatic l-amino acid decarboxylase (AADC) is target of autoantibodies in autoimmune polyendocrin... more 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. 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. 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. 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.
Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5-phosphate (PLP... more Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe 152 variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe 152 variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400fold lower. Thus, Phe 152 is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp 108 , as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.
The vitamin B 6-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a larg... more The vitamin B 6-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in amino acid metabolism. These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and protein structural organization. Almost 1.5% of all genes in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of enzyme-catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), serine hydroxymethyltransferase (tumors and malaria), ornithine decarboxylase (African sleeping sickness and, potentially, tumors), alanine racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or metabolic pathway, the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and ongoing research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.
The effect of N-(5'-phosphopyridoxyl)-L-5-hydroxytryptophan, N-(5'-phosphopyridox... more The effect of N-(5'-phosphopyridoxyl)-L-5-hydroxytryptophan, N-(5'-phosphopyridoxyl)-D-5-hydroxytryptophan and N-(5'-phosphopyridoxyl)-5-hydroxytryptamine on the reactivation of apoDopa decarboxylase to holoenzyme has been investigated. The different degree of inhibition exerted by these adducts has been interpreted on the basis of a different orientation of the 2 isomers of 5-HTP at the active of Dopa decarboxylase.
The unusual oxygen-consuming oxidative deamination reaction catalyzed by the pyridoxal 5′-phospha... more 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.
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Papers by C. Voltattorni