Cloning, nucleotide sequence, and expression of a p-hydroxybenzoate hydroxylase isozyme gene from Pseudomonas fluorescens.

Abstract A gene encoding for a putative isozyme of p-hydroxy-benzoate hydroxylase (PHBH) has been isolated from Pseudomonas fluorescens (ATCC 13525). A comparison of the translated amino acid sequence with that of the known PHBH from P. fluorescens revealed that the new enzyme contains 3 additional amino acids and has 73% absolute homology to the previously known enzyme; conservation of secondary and active-site structures implied that the isozyme and known enzyme share the same general tertiary structure. Subsequent expression of the isozyme in Escherichia coli produced an enzyme with a specific activity about half that of the previously characterized PHBHs from P. fluorescens and Pseudomonas aeruginosa; in addition, somewhat weaker binding affinities for both NADPH and p-hydroxybenzoate were observed. Speculations are made on the reason for the existence of the isozyme, which does not appear to be expressed routinely in P. fluorescens.