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

• Published in: The Journal of biological chemistry 1993-08, Vol.268 (23), p.17057-17062
• Main Authors: SHUMAN, B, DIX, T. A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anaerobiosis; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Cloning, Molecular; DNA, Bacterial - isolation & purification; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - isolation & purification; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Oxidoreductases; Polymerase Chain Reaction; Protein Biosynthesis; Pseudomonas fluorescens; Pseudomonas fluorescens - enzymology; Pseudomonas fluorescens - genetics; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)85301-2

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.