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 b y five consecutive steps to apparent homogeneity. The enrichment was 5 0-fold with a yield of about 20 %. The enzyme is a homodimeric flavopr otein 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-hydroxyb enzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-fluoro-4-hydr oxybenzoate, 2-chloro-4-hydroxybenzoate, and 2,4-dihydroxybenzoate. Wi th all aromatic substrates, no uncoupling of hydroxylation was observe d. The gene encoding 4-hydroxybenzoate hydroxylase from Pseudomonas sp . CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis re vealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence res embling an E. coli promotor was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The deduced amino acid s equence of Pseudomonas sp. CBS3 4-hydroxybenzoate hydroxylase revealed 53 % identity with that of the pobA enzyme from Pseudomonas fluoresce ns for which a three-dimensional structure is known. The active-site r esidues 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 mutage nesis data of 4-hydroxybenzoate hydroxylase from P. fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specif icity.