Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum.

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH(2)PO(4) and K(2)HPO(4) as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 A on a synchrotron beamline. The crystal belongs to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 94.72, c = 359.68 A, gamma = 120 degrees . The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 A(3) Da(-1). The structure was solved by molecular replacement. Structure refinement is in progress.

Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover.

Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln. This mutation removes a negative charge from a conserved buried charge pair. The properties of the Glu49Gln mutant enzyme are consistent with increased positive potential in the active site, but the mutant enzyme is difficult to study because it is unstable. There are two important changes in the catalytic function of the mutant enzyme as compared to the wild-type. First, the rate of hydroxylation of p-hydroxybenzoate by the transiently formed flavin hydroperoxide is an order of magnitude faster than in the wild-type. This result is consistent with one function proposed for the positive potential in the active site-to stabilize the negative C-4a-flavin alkoxide leaving group upon heterolytic fission of the peroxide bond. However, the mutant enzyme is a poorer catalyst than the wild-type enzyme because (unlike wild-type) the binding of p-hydroxybenzoate is a rate-limiting process. Our analysis shows that the mutant enzyme is slow to interconvert between conformations required to bind and release substrate. We conclude that the new open structure found in crystals of the Arg220Gln mutant enzyme [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613] is integral to the process of binding and release of substrate from oxidized enzyme during catalysis.

Purification and properties of p-hydroxybenzoate hydroxylases from Rhodococcus strains.

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl- and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions.

4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3. Purification, characterization, gene cloning, sequence analysis and assignment of structural features determining the coenzyme specificity.

4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50-fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44-kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4-hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45 degrees C. Chloride ions were inhibitory, and competitive with respect to NADH. 4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-fluoro-4-hydroxybenzoate, 2-chloro-4-hydroxybenzoate, and 2,4-dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed. The gene encoding 4-hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The deduced amino acid sequence of Pseudomonas sp. CBS3 4-hydroxybenzoate hydroxylase revealed 53% identity with that of the pobA enzyme from Pseudomonas fluorescens for which a three-dimensional structure is known. The active-site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three-dimensional fold. Based on an isolated region of sequence divergence and site-directed mutagenesis data of 4-hydroxybenzoate hydroxylase from P. fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from a Klebsiella pneumoniae mutant strain.

Unlike the parent wild-type strain, the Klebsiella pneumoniae mutant strain MAO4 has a 4-HBA+ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when the Acinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoate K. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.

Purification and characterization of Acinetobacter calcoaceticus 4-hydroxybenzoate 3-hydroxylase after its overexpression in Escherichia coli.

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.

Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa.

The monooxygenase, p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) has been isolated and purified from Pseudomonas aeruginosa. The reaction catalysed is linked to the pathways for degradation of aromatic compounds by microorganisms. The enzyme has been quantitatively characterized in this paper for use in the mechanistic analysis of the protein by site-directed mutagenesis. This can be achieved when the results presented are used in combination with the information on the sequence and structure of the gene for this protein and the high-resolution crystallographic data for the protein from P. fluorescens. The protein is a dimer of identical sub-units in solution, and has one FAD per polypeptide with a monomeric molecular weight of 45,000. A full steady-state kinetic analysis was carried out at the optimum pH (8.0). A Vmax of 3750 min-1 at 25 degrees C was calculated, and the enzyme has a concerted-substitution mechanism, involving the substrates, NADPH, oxygen, and p-hydroxybenzoate. Extensive analyses of the reactions of reduced enzyme with oxygen were carried out. The quality of the data obtained confirmed the mechanisms of these reactions as proposed earlier by the authors for the enzyme from P. fluorescens. It was found that the amino acid residue differences between enzyme from P. fluorescence and aeruginosa do marginally change some observed transient state kinetic parameters, even though the structure of the enzyme shows they have no direct role in catalysis. Thus, transient state kinetic analysis is an excellent tool to examine the role of amino acid residues in catalysis.

The temperature and pH dependence of some properties of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The free and complexed flavoprotein, p-hydroxybenzoate hydroxylase, was studied by light-absorption, circular-dichroism and fluorescence techniques as a function of the pH. The following compounds served as ligands for the enzyme: p-hydroxybenzoate, p-fluorobenzoate, benzoate, p-aminobenzoate and tetrafluoro-p-hydroxybenzoate. Depending on the technique used, the various ligands exhibit pH-dependent physical properties and dissociation constants. The data can be fitted with pKa values in the range 7.7-7.9. It is suggested that this pKa value belongs to a tyrosine residue in the active center of the enzyme. This assignment is supported by published data and additional experiments.

The influence of purification and protein heterogeneity on the crystallization of p-hydroxybenzoate hydroxylase.

The structure of the enzyme p-hydroxybenzoate hydroxylase was determined to a resolution of 0.25 nm [Wierenga et al. (1979) J. Mol. Biol. 131, 53-73] with crystals belonging to space group C222(1). Subsequently it was impossible to repeat the growth of this crystal form and only poor quality tetragonal crystals could be obtained. We have thoroughly investigated this problem and found that Cibacron-blue-purified enzyme appears to be heterogeneous with respect to aggregation state and Cys-116 oxidation. Most importantly, it could be firmly established that C222(1) crystals can only be grown from purely dimeric p-hydroxybenzoate hydroxylase possessing an intact SH group. Ion-exchange chromatography on DEAE-Sepharose can successfully remove those forms of the enzyme which impede successful crystallization. Sulfite and dithiothreitol improve crystallization by dissociating the enzyme oligomers into dimers; sulfite especially gives excellent results.

Chemical modification of tyrosine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: assignment in sequence and catalytic involvement.

p-Hydroxybenzoate hydroxylase was modified by diethyl pyrocarbonate at pH values greater than 7 and by p-diazobenzoate. Modification of the enzyme by diethyl pyrocarbonate abolishes the affinity of the enzyme for the substrate p-hydroxybenzoate. Modification by p-diazobenzoate has the same effect on the enzyme. The enzyme is protected against these modifications by the effector p-fluorobenzoate. The data indicate that the modification of one tyrosine residue in the active center of the enzyme is responsible for the loss of enzyme activity. This tyrosine residue has been identified by sequence studies using radioactively labeled p-diazobenzoate and was found to be most probably Tyr-222. Diethyl pyrocarbonate reacts with a tyrosine residue in the active center other than Tyr-222; the former could not be identified. Sequence studies further showed that Cys-211 is also partially modified by p-diazobenzoate. In addition, the sequence of residues 343-345 was found to be Ser-Trp-Trp instead of the tentative assignment Ser-Tyr-Trp made earlier. The results are briefly discussed on the basis of the existing three-dimensional model of the enzyme.

Purification and properties of NADH/NADPH-dependent p-hydroxybenzoate hydroxylase from Corynebacterium cyclohexanicum.

Crude soluble extracts of Corynebacterium cyclohexanicum, grown on cyclohexanecarboxylic acid, were found to contain 4-hydroxybenzoate 3-hydroxylase which functions with NADH as well as NADPH. The purified enzyme preparation was electrophoretically homogeneous and contained FAD as prosthetic group. The relative molecular mass of the enzyme was estimated to be about 47000 by native and denaturated acrylamide gel electrophoresis, indicating that it is monomeric. The enzyme was stable at 60 degrees C for 10 min. The enzyme was highly specific for p-hydroxybenzoate. The activity was inhibited by several aromatic analogues of p-hydroxybenzoate such as p-aminobenzoate, p-fluorobenzoate, o-hydroxybenzoate, m-hydroxybenzoate, 2,4-dihydroxygenzoate, and 2,5-dihydroxybenzoate. The Km value for NADH was fairly constant, about 45 microM, in the pH range 7.0-8.4, whereas the Km value for NADPH increased from 63 microM to 170 microM as the pH rose from 7.0 to 8.4. V values in the same pH range, however, were approximately constant in both cases; about 30 mumol min-1 mg-1 for NADH, and 26 mumol min-1 mg-1 for NADPH. Mg2+ was required for full activity of the enzyme in low concentrations of phosphate buffer. The enzyme was inhibited by C1- which was non-competitive with respect to NADH, NADPH and p-hydroxybenzoate.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 2. Fitting of the amino-acid sequence to the tertiary structure.

The complete primary and tertiary structure of p-hydroxybenzoate hydroxylase is now known. The amino acid sequences of the two largest CNBr peptides have been fitted to the electron-density map at 0.25-nm resolution. The parts of the polypeptide chain contributing the residues to the FAD-binding site and the residues of the substrate-binding site have been identified. The active site is located in a large hydrophobic area enclosed by all domains of the enzyme structure. Here the substrate, p-hydroxybenzoate, is bound near, but not in direct contact with, the isoalloxazine ring system of FAD. Many side chains from the C-terminal part of the polypeptide chain are involved in subunit-subunit interactions. In the center of one of the largely hydrophobic contact areas between the subunits, a cluster of six aromatic amino acids was found.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 1. Completion of the elucidation of the primary structure.

As a final step in the elucidation of the primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, the amino acid sequences of a CNBr peptide (CB1, positions 111-276), that accounts for the middle part of the sequence, and the C-terminal CNBr peptide (CB2, positions 277-394) from the enzyme were determined. Important sequence information was obtained from two subfragments that were formed by the cleavage with CNBr of the Met-Thr sequence (positions 346-347) in peptide CB2. The alignment of the two subfragments from peptide CB2 and three one-residue overlaps between peptides from one of these subfragments were confirmed by investigation of well-resolved parts of a 0.25-nm electron-density map. The sequence of residues 343-346 could not be determined with chemical methods and was assigned from the size and shape of the amino acids in the electron-density map. An important tool in the analysis of the amino acid sequence of peptide CB1 was the proteinase Lys-C from Lysobacter enzymogenes, which preferentially cleaves at lysine residues.

A study of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Improved purification, relative molecular mass, and amino acid composition.

The purification procedure for p-hydroxybenzoate hydroxylase has been modified by replacement of the DEAE-cellulose (DE-32) column in the original procedure by a Sephadex--Cibacron-blue affinity column. In this way the yield of enzyme could be improved from 16% to about 40--50%. Preparative gel chromatography indicated that the enzyme does not exist as a monomeric species as earlier believed but mainly as a dimer. Sodium dodecyl sulfate gel electrophoresis of purified enzyme revealed a minimum relative molecular mass (Mr) of 43000--45000. Analytical gel chromatography, sedimentation equilibrium and sedimentation velocity experiments showed that the enzyme exists in solution mainly as a dimer but also in higher-order quaternary structures (presumably tetramer and hexamer). Temperature dependence of the distribution of the oligomers suggests that the association is of hydrophobic nature. The amino acid composition of the enzyme is also presented. The enzyme contains no disulfide but five sulfhydryl groups. In the native state of the enzyme only one sulfhydryl group is accessible to N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid). The iso-electric point of the enzyme was found to be 5.8.