Primary and tertiary structure studies of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Isolation and alignment of the CNBr peptides; interactions of the protein with flavin adenine dinucleotide.
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p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a beta alpha beta unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter. (+info)
The amino-acid sequence of the three smallest CNBr peptides from p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.
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After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: (Formula: See Text). (+info)
Inhibition of p-hydroxybenzoate hydroxylase by anions: possible existence of two anion-binding sites in the site for reduced nicotinamide adenine dinucleotide phosphate.
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Certain anions were found to inhibit p-hydroxybenzoate hydroxylase from Pseudomonas desmolytica. The inhibition was of competitive or mixed type with respect to NADPH (apparent Ki = 4-30 mM). Among the anions, monovalent anions such as halogen ions and azide inhibited ionization of the phenolic hydroxyl group of the substrate (p-hydroxybenzoate) on binding with the enzyme . substrate complex of p-hydroxybenzoate hydroxylase, without dissociating the substrate from the enzyme. On the other hand, multivalent anions (anions of polybasic acids), such as inorganic phosphate, borate, and sulfate, did not inhibit the ionization. Halogen ions induced remarkable spectral changes in the FAD moiety of the enzyme on binding, while the change due to inorganic phosphate was only slight. Chloride inhibited the binding of NADH with the enzyme as well as that of NADPH, whereas borate inhibited the binding of only NADPH. These results indicate that the monovalent and multivalent anions probably bind to the sites in the enzyme which interact, respectively, with the pyrophosphate and 2'-phosphate moieties of NADPH. The results provide strong support for the catalytic mechanism in which the phenolate anion of p-hydroxybenzoate participates in the process of substrate hydroxylation by C (4a) peroxyflavin. The results also suggest that repeated ionization/neutralization of the phenolic hydroxyl group of the substrate may occur during one cycle of the catalytic turnover. (+info)
Reaction of 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase with hydrogen peroxide. Formation of a covalent flavin-protein linkage.
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Hydrogen peroxide reacts with 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase to yield a long wavelength intermediate (lambda max = 360, 620 nm) which can be isolated in stable form on removal of excess H2O2. The blue flavin derivative slowly decays in a second peroxide-dependent reaction to yield a new flavin product lacking long wavelength absorbance (lambda max = 408, 472 nm). This final peroxide-modified enzyme binds p-hydroxybenzoate with a 10-fold lower affinity than does the native enzyme; furthermore, substrate binding leads to the inhibition of enzyme reduction by NADPH. Trichloroacetic acid treatment of the final peroxide-modified enzyme results in the quantitative conversion of the bound flavin to free FAD. However, gel filtration of the modified enzyme in guanidine hydrochloride at neutral pH leads to the co-elution of protein and modified flavin. The nondenatured peroxide product reacts rapidly with hydroxylamine to yield 2-NHOH-substituted FAD. These observations indicate that the secondary reaction of peroxide with the blue intermediate from 2-thio-FAD p-hydroxybenzoate hydroxylase results in the formation of an acid-labile covalent flavin-protein linkage within the enzyme active site, involving the flavin C-2 position. (+info)
A histidine residue in p-hydroxybenzoate hydroxylase essential for binding of reduced nicotinamide adenine dinucleotide phosphate.
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Chemical modification with diethylpyrocarbonate (ethoxyformic anhydride) was examined to demonstrate the existence of an essential histidine residue at the NADPH-binding site of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas desmolytica. Among some ligands, NADPH was noticeable in protecting the enzyme from the modification-caused inactivation. Although several amino acid residues were modified during the inactivation process, inhibition of the enzyme could be correlated with modification of a single histidine residue which was masked by addition of NADPH. The pK of the essential histidine residue was estimated to be 6.5-6.7. The Kd (Km) for NADPH of the inactivated enzyme was shown to have been increased greatly, although the Kd for substrate (p-hydroxybenzoate) was not changed. (+info)
Photodehalogenation of 7- and 8-halogen-substituted flavins. Photochemistry of the reduced flavin chromophore.
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Flavodoxin was reconstituted with 8-chloro- and 7-bromo-FMN and p-hydroxybenzoate hydroxylase with the analogous FAD derivatives. In all cases, the spectral properties of the artificial enzymes changed as a result of photoreduction in the presence of ethylenediaminetetraacetate or oxalate as sources of reducing equivalents. The same changes were found to occur on irradiation of the enzymes which had been reduced previously in the dark under anaerobic conditions with dithionite. Using analogous 7- and 8-chlorolumiflavins, the observed changes were shown to be due to a novel photoreaction of the reduced flavin chromophore, in which either the 7- or 8-halogen substituent is eliminated and replaced by a proton derived from the solvent. The same reaction was shown to occur with 7,8-bis-norlumiflavin where 1 deuterium atom was incorporated into the molecule as a result of photoirradiation of the reduced flavin in deuterated medium. In the case of p-hydroxybenzoate hydroxylase, both the 8-chloro-FAD and 7-bromo-FAD enzymes, as well as their 8-nor-FAD and 7-nor-FAD photoproducts, possessed catalytic activity comparable to that of the native enzyme. (+info)
An essential arginine residue at the substrate-binding site of p-hydroxybenzoate hydroxylase.
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p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) was rapidly inactivated by treatment with phenylglyoxal, by a process obeying pseudo-first order kinetics. The reaction with the reagent was also examined by amino acid analyses, radioactivity measurements, and spectrophotometric analyses. Results of these analyses were consistent with each other, which shows that the inactivation was due to modification of argiine residue(s). Addition of saturating amounts of p-hydroxybenzoate (or benzoate) during the treatment resulted in marked protection of the enzyme from the inactivation as well as a significant decrease in modification of arginine residues, while phenol showed no effect. Modification in the absence of p-hydroxybenzoate caused a spectral change in the flavin moiety of the enzyme similar to that due to the enzyme.substrate complex formation, and losses in both the overall activity and the substrate-binding ability accompanied the spectral change. On the other hand, such spectral change was not observed and the substrate-binding ability was retained even after the overall activity had decreased to a great extent when p-hydroxybenzoate as added during the modification treatment. These results suggest that phenylglyoxal (an analogue of the substrate) was incorporatd into the substrate-binding site and that an arginine residue is involved in the site, having an interaction with the carboxylate anion of the substrate. (+info)
Purification and characterization of Acinetobacter calcoaceticus 4-hydroxybenzoate 3-hydroxylase after its overexpression in Escherichia coli.
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4-Hydroxybenzoate 3-hydroxylase [EC 1.14.13.2] from Acinetobacter calcoaceticus was purified to homogeneity following the 40-fold overexpression of this gene (pobA) in Escherichia coli. Overexpression was accomplished by placing the folA gene (encoding trimethoprim-resistant dihydrofolate reductase) directly downstream of the pobA gene, and demanding growth of recombinants on elevated concentration of trimethoprim. Presumably, the surviving variants have undergone a genetic alteration which allowed the overexpression of both folA and pobA. 4-Hydroxybenzoate 3-hydroxylase was purified in two chromatographic steps, characterized biochemically, and its properties were compared to those of its homolog from Pseudomonas fluorescens. The two enzymes differ in their response to Cl- ion inhibition. A single amino acid change in the putative NADPH-binding site is proposed to account for this difference. The inhibitory and catalytic properties of substrate analogs were also examined. (+info)