Affinity labeling of pig kidney 3,4‐dihydroxyphenylalanine (Dopa) decarboxylase with<i>N</i>‐(bromoacetyl)pyridoxamine 5′‐phosphate

Dominici, Paola; Maras, Bruno; Mei, Giampiero; Voltattorni, Carla Borri

doi:10.1111/j.1432-1033.1991.tb16296.x

Cited by 9 publications

(5 citation statements)

References 18 publications

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“…This PLP emission is probably due to the occurrence of energy transfer from tryptophan residue(s) to the reduced coenzyme. A similar process has been observed already for N-(bromoacetyl)pyridoxamine 5'-phosphate-modified apoenzyme (Dominici et al, 1991a). The intrinsic emission fluorescence spectra of reduced holo-DDC in GuCl solutions up to 6 M are characterized by changes (red shift of the 326 nm emission and a decrease in the emission intensity) identical to those observed for the apoenzyme.…”

Section: Binding Of Coenzymesupporting

confidence: 80%

Dissociation, unfolding and refolding trials of pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase

Dominici

Moore

Voltattorni

1993

Biochemical Journal

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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 studied. Attempts at refolding resulted in the findings that: (1) partial reactivation is observed only starting from enzyme denatured at concentrations below 1.5 M GuCl, and (2) starting from completely denatured protein, the refolding process is apparently reversible down to concentrations of approx. 2 M GuCl. Taken together, this would seem to indicate that the monomer-dimer transition is impaired under the experimental conditions tested. A plausible model is presented for the unfolding/refolding of pkDDC.

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Section: Binding Of Coenzymesupporting

confidence: 80%

Dissociation, unfolding and refolding trials of pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase

Dominici

Moore

Voltattorni

1993

Biochemical Journal

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“…An arginine residue plays an essential role in substrate binding4 and the modification of an unidentified histidine residue with diethylpyrocarbonate abolishes the enzyme activity 5. Inactivation by iodoacetamide suggests a role for the cysteine C4386 and the cysteine C111 may lie in the binding site of AADC 7. Previous extensive mutation analysis performed on the AADC has highlighted the importance of numerous residues8–10 without indicating their role in the function or the stability of the protein.…”

Section: Introductionmentioning

confidence: 99%

Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5´-phosphate-dependent decarboxylase: A functional study

et al. 1999

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The pyridoxal-5Ј-phosphate-dependent enzymes (B 6 enzymes) are grouped into three main families named ␣, ␤, and ␥. Proteins in the ␣ and ␥ families share the same fold and might be distantly related, while those in the ␤ family exhibit specific structural features. The rat aromatic L-amino acid decarboxylase (AADC; EC(4.1.1.28)) catalyzes the synthesis of two important neurotransmitters: dopamine and serotonin. It binds the cofactor pyridoxal-5Ј-phosphate and belongs to the ␣ family. Despite the low level of sequence identity (approximately 10%) shared by the rat AADC and the sequences of the enzymes belonging to the B 6 enzymes family, including the known three-dimensional structures, a multiple sequence alignment was deduced. A model was built using segments belonging to seven of the eleven known structures. By homology, and based on knowledge of the biochemistry of the aspartate aminotransferase, structurally and functionally important residues were identified in the rat AADC. Site-directed mutagenesis of the conserved residues D271, T246, and C311 was carried out in order to confirm our predictions and highlight their functional role. Mutation of D271A and D271N resulted in complete loss of enzyme activity, while the D271E mutant exhibited 2% of the wild-type activity. Substitution of T246A resulted in 5% of the wild-type activity while the C311A mutant conserved 42% of the wild-type activity. A functional model of the AADC is discussed in view of the structural model and the complementary mutagenesis and labelling studies. Proteins 1999;37:191-203.

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“…in the reaction mechanism [8][9][10]. The complete primary structure has been determined by Edman degradation [11] and crystallographic studies are in progress [12].…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes

Moore

Dominici

Voltattorni

1996

Biochemical Journal

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L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To advance structure-function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the later protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the Vmax was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme-apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour.

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Affinity labeling of pig kidney 3,4‐dihydroxyphenylalanine (Dopa) decarboxylase withN‐(bromoacetyl)pyridoxamine 5′‐phosphate

Cited by 9 publications

References 18 publications

Dissociation, unfolding and refolding trials of pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase

Dissociation, unfolding and refolding trials of pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase

Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5´-phosphate-dependent decarboxylase: A functional study

Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes

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