CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF A PARA-HYDROXYBENZOATE HYDROXYLASE ISOZYME GENE FROM PSEUDOMONAS-FLUORESCENS

A gene encoding for a putative isozyme of p-hydroxybenzoate hydroxylas e (PHBH) has been isolated from Pseudomonas fluorescens (ATCC 13525). A comparison of the translated amino acid sequence with that of the kn own PHBH from P. fluorescens revealed that the new enzyme contains 3 a dditional amino acids and has 73% absolute homology to the previously known enzyme; conservation of secondary and active-site structures imp lied that the isozyme and known enzyme share the same general tertiary structure. Subsequent expression of the isozyme in Escherichia coli p roduced an enzyme with a specific activity about half that of the prev iously characterized PHBHs from P. fluorescens and Pseudomonas aerugin osa; in addition, somewhat weaker binding affinities for both NADPH an d p-hydroxybenzoate were observed. Speculations are made on the reason for the existence of the isozyme, which does not appear to be express ed routinely in P. fluorescens.