4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3

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Abstract
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°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 promotor 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.