4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3

Abstract

Technical University Hamburg-Harburg, Hamburg, Germany 4-Hydroxybenzoate hydroxylase from Pseudomonus 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°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-tluoro-4-hydroxybenzoate, 2-cliloro-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 11 82 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promotor was identified, which led to constitutive expression of the cloned gene in E. coli TG1 . The deduced amino acid sequence of Pseudomanus sp. CBS3 4-hydroxybenzoate hydroxylase revealed 53 % identity with that of the pobA enzyme from Pseudomonasfluorescens 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 f? ,fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.

Aerobic Metabolism of 4-Hydroxybenzoic Acid in Archaea via an Unusual Pathway Involving an Intramolecular Migration (NIH Shift)

“…Induction of GDO was demonstrated in extracts from 4HBA-grown cells, along with stoichiometric accumulation of GA from 4HBA in biotransformation experiments. In contrast, most bacteria aerobically metabolize 4HBA by 3-hydroxylation to yield protocatechuate, which serves as the ring cleavage substrate (25,28,34,35,38,39,55,60,62,64). Metabolism of 4HBA by decarboxylation to phenol, followed by ortho hydroxylation to catechol, has also been suggested in Rhodococcus opacus (35).…”

Interdomain binding of NADPH in p-Hydroxybenzoate Hydroxylase as Suggested by Kinetic, Crystallographic and Modeling Studies of Histidine 162 and Arginine 269 Variants

“…Dissociation constants of enzyme-ligand complexes were determined fluorimetrically (31). Oxygen consumption experiments were performed as described previously (3). Aromatic product formation was analyzed by reverse phase HPLC (29).…”

“…The dihydroxylated aromatic product is readily subject to ring fission and further catabolism, allowing the microbes to grow (2). p-Hydroxybenzoate hydroxylase has been isolated from many microorganisms, and several gene sequences are presently known (3). However, most information regarding its structure and function comes from studies on the strictly NADPH-dependent enzymes from Pseudomonas strains (4).…”

“…Despite their location near the protein surface (Fig. 1), both residues are highly conserved in p-hydroxybenzoate hydroxylase enzymes of known primary structure (3).…”

Gene Cluster on pAO1 of Arthrobacter nicotinovorans Involved in Degradation of the Plant Alkaloid Nicotine: Cloning, Purification, and Characterization of 2,6-Dihydroxypyridine 3-Hydroxylase

“…In the third fingerprint motif of this family GDAAH, the H residue is replaced in 2,6-DHPH by V. Despite these alterations in residues shown to be important for cofactor binding, FAD is nevertheless tightly bound by the enzyme. There are several FAD-dependent hydroxylases known, with a loosely bound flavin cofactor, like 4-hydroxybenzoate hydroxylase (33). However, this enzyme shows the same conserved amino acid sequences as those with tightly bound FAD (11,12).…”