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Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes
No full textL-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
Chemical modification of pig kidney 3,4-dihydroxyphenylalanine decarboxylase with diethyl pyrocarbonate. Evidence for an essential histidyl residue
No full textDiethyl pyrocarbonate inhibits pig kidney holo-3,4-dihydroxyphenylalanine decarboxylase with a second-order rate constant of 1170 M-1 min-1 at pH 6.8 and 25 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity can be restored by hydroxylamine, and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.03. Complete inactivation of 3,4-dihydroxyphenylalanine decarboxylase requires the modification of 6 histidine residues/mol of enzyme. Statistical analysis of the residual enzyme activity and of the extent of modification shows that, among 6 modifiable residues, only one is critical for activity. Protection exerted by substrate analogues, which bind to the active site of the enzyme, suggests that the modification occurs at or near the active site. The modified inactivated 3,4-dihydroxyphenylalanine decarboxylase still retains most of its ability to bind substrates. Thus, it may be suggested that the inactivation of enzyme by diethyl pyrocarbonate is not due to nonspecific steric or conformational changes which prevent substrate binding. However, the modified enzyme fails to produce at high pH either an enzyme-substrate complex or an enzyme-product complex absorbing at 390 nm. Considerations on this peculiar feature of the modified enzyme consistent with a catalytic role for the modified histidyl residue are discussed. The overall conclusion of this study may be that the modification of only one histidyl residue of 3,4-dihydroxyphenylalanine decarboxylase inactivates the enzyme and that this residue plays an essential role in the mechanism of action of the enzyme
Interaction of aromatic amino acids in D and L forms with 3,4-dihydroxyphenylalanine decarboxylase from pig kidney
No full textInteraction of aromatic amino acids in D and L forms with 3,4-dihydroxyphenylalanine decarboxylase from pig kidne
Transaldimination induces coenzyme reorientation in pig kidney Dopa decarboxylase
No full textTransaldimination, coenzyme orientatio
Stereospecificity of sodium borohydride reduction of pig kidney dopa decarboxylase
No full textSodium boro[3H]hydride reduction of pig kidney 3,4 dihydroxyphenylalanine decarboxylase followed by complete hydrolysis of the enzyme produced epsilon-[3H]pyridoxyllysine. Degradation of this material to 4'-[3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase showed that the re side at C-4' of the coenzyme is exposed to solvent. In order to determine the face exposed to the solvent in the external Schiff's base, attempts to trap reaction intermediates were made by reduction with sodium boro [3H]hydride of the holoenzyme in the presence of various substrates or substrate analogs. In all cases, covalently bound radioactive material was found which was identified as epsilon-N-pyridoxyllysine. These results suggest that the internal Schiff's base is in mobile equilibrium with the external Schiff's base and that sodium borohydride reduction displaces this equilibrium, resulting in complete reduction of the internal Schiff's base
MODIFIED PURIFICATION OF L-AROMATIC AMINO-ACID DECARBOXYLASE FROM PIG KIDNEY
No full textMODIFIED PURIFICATION OF DOPA DECARBOXYLASE FROM PIG KIDNE
Dissociation, unfolding and refolding trials of pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase
No full textThe 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
Characterization of putative PLP-dependent beta C-S lyase from Corynebacterium diphtheriae, a possible target for a new antimicrobial agent.
No full textThe continuous emergence of antibiotic resistance in microbial pathogens requires a sustained effort to identify new antimicrobial compounds and targets. The biosynthesis of methionine is an attractive target given its importance in protein and DNA metabolism. Moreover, most of the steps in this pathway are absent in mammals, lessening the opportunity of unwanted side effects.
Herein, detailed biochemical characterization of a putative pyridoxal 5’-phosphate (PLP)-dependent beta C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in Escherichia coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters and ligand-induced spectral transitions of the recombinant protein by a combination of UV/Vis and fluorescence spectroscopy.
The 3D structure of beta C-S lyase from Corynebacterium diphtheriae has already been solved at 1.99 Å resolution (Joint Center for Structural Genomics). The enzyme is a homodimer composed of ~42 kDa subunits, each associated with one molecule of PLP. Structural comparison of beta C-S lyase from Corynebacterium diphtheriae with beta C-S lyase from Streptococcus anginosus1 and cystalysin from Treponema denticola2 indicates a similarity in overall folding and active site residues. We used site-directed mutagenesis to highlight the importance of the active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties and spectra of enzyme-ligand complexes.
Better understanding of the active site of Corynebacterium diphtheriae beta C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors, as antibacterial therapeutics
Affinity labelling of pig kidney Dopa decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active site cysteine
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