Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Dominici, Paola; Moore, Patrick S.; Castellani, Silvia; Bertoldi, Mariarita; Voltattorni, Carla Borri

doi:10.1002/pro.5560060921

Cited by 25 publications

(18 citation statements)

References 40 publications

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“…Titration analysis of the wild-type, G102S, F309L, S147R, and A275T in the apo form with PLP, and fitting of the data to the appropriate equation yielded the K D(PLP) values reported in Table 1. The data indicate that the PLP binding affinity of human wild-type DDC is equal to that of pig kidney DDC previously measured (Dominici et al 1997), and not remarkably affected by replacement of Gly102 by Ser. In contrast, substitutions of Phe309 by Leu, Ser147 by Arg, and Ala275 by Thr, cause a~8-, 19-, and 170-fold decrease in PLP binding affinity, respectively.…”

Section: Resultsmentioning

confidence: 55%

Molecular insights into the pathogenicity of variants associated with the aromatic amino acid decarboxylase deficiency

Montioli

Cellini

Voltattorni

2011

J of Inher Metab Disea

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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.

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Section: Resultsmentioning

confidence: 55%

Molecular insights into the pathogenicity of variants associated with the aromatic amino acid decarboxylase deficiency

Montioli

Cellini

Voltattorni

2011

J of Inher Metab Disea

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“…The mutation lies within residues 64 to 155 that is highly conserved within all the published vertebrate amino acid sequences. 11,12 Within this area, residues 68 to 105 form an ␣ helix that runs along the face between the two subunits (Fig 2). This region is absent from a naturally occurring differentially spliced form of the AADC transcript *Aromatic L-amino acid decarboxylase plasma activities and wild type and mutant recombinant protein activities were determined by measurement of dopamine production following incubation with levodopa, HPLC separation and electrochemical detection, essentially as previously described.…”

Section: Discussionmentioning

confidence: 99%

Levodopa‐responsive aromatic L–amino acid decarboxylase deficiency

Chang

Sharma

Marsh

et al. 2004

Annals of Neurology

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We report three siblings, who were treated empirically with levodopa combined with carbidopa. There was an immediate therapeutic response. Biochemical investigation surprisingly showed the clinical phenotype to be caused by aromatic L-amino acid decarboxylase deficiency. Molecular characterization showed a homozygous point mutation (c.387 G-->A) in exon 3. Kinetic studies showed the mutation to decrease the binding affinity for the substrate. This, combined with structural modeling suggesting alteration of active site configuration, provided an explanation for the therapeutic response to levodopa.

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“…Although these results, in contrast with those previously reported [3], would rule out the idea that during the decarboxylation of -dopa a decarboxylation-dependent transamination occurs, they do not explain the inactivation process. Because the binding constant, K d , of recombinant DDC for PLP has been estimated to be 44 nM [14], at the enzyme concentration used by us and by O'Leary and Baughn to assay decarboxylase activity we would expect dissociation of the coenzyme from the enzyme. The following experiment confirmed this idea.…”

Section: Figure 1 Coenzyme Content and Decarboxylase Activity During mentioning

confidence: 98%

Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions

Bertoldi

Voltattorni

2000

Biochemical Journal

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Analysis of the reaction of dopa decarboxylase (DDC) with L-dopa reveals that loss of decarboxylase activity with time is observed at enzyme concentrations approximately equal to the binding constant, Kd, of the enzyme for pyridoxal 5ƀ-phosphate (PLP). Instead, at enzyme concentrations higher than Kd the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5ƀ-phosphate (PMP) is formed during the reaction and that dissociation of coenzyme occurs at low enzyme concentration, leading to the formation of a PLP-L-dopa Pictet–Spengler cyclic adduct. Taken together, these results indicate that decarboxylation-dependent transamination does not accompany the decarboxylation of L-dopa proposed previously [O'Leary and Baughn (1977) J. Biol. Chem. 252, 7168–7173]. Nevertheless, when the reaction of DDC with L-dopa is studied under anaerobic conditions at an enzyme concentration higher than Kd, we observe that (1) the enzyme is gradually inactivated and inactivation is associated with PMP formation and (2) the initial velocity of decarboxylation is approximately half of that in the presence of O2. Similar behaviour is observed by comparing the reaction with L-5-hydroxytryptophan occurring in aerobiosis or in anaerobiosis. Therefore the reaction of DDC with L-aromatic amino acids seems to be under O2 control. In contrast, the reactivity of the enzyme with D-aromatic amino acids does not change in the presence or absence of O2. These and other results, together with previous results on the effect exerted by O2 on reaction specificity of DDC towards aromatic amines [Bertoldi, Frigeri, Paci and Borri Voltattorni (1999) J. Biol. Chem. 274, 5514–5521], suggest a productive effect of O2 on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines.

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Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Cited by 25 publications

References 40 publications

Molecular insights into the pathogenicity of variants associated with the aromatic amino acid decarboxylase deficiency

Molecular insights into the pathogenicity of variants associated with the aromatic amino acid decarboxylase deficiency

Levodopa‐responsive aromatic L–amino acid decarboxylase deficiency

Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions

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