Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa.
The monooxygenase, p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) has been isolated and purified from Pseudomonas aeruginosa. The reaction catalysed is linked to the pathways for degradation of aromatic compounds by microorganisms. The enzyme has been quantitatively characterized in this paper for use in the mechanistic analysis of the protein by site-directed mutagenesis. This can be achieved when the results presented are used in combination with the information on the sequence and structure of the gene for this protein and the high-resolution crystallographic data for the protein from P. fluorescens. The protein is a dimer of identical sub-units in solution, and has one FAD per polypeptide with a monomeric molecular weight of 45,000. A full steady-state kinetic analysis was carried out at the optimum pH (8.0). A Vmax of 3750 min-1 at 25 degrees C was calculated, and the enzyme has a concerted-substitution mechanism, involving the substrates, NADPH, oxygen, and p-hydroxybenzoate. Extensive analyses of the reactions of reduced enzyme with oxygen were carried out. The quality of the data obtained confirmed the mechanisms of these reactions as proposed earlier by the authors for the enzyme from P. fluorescens. It was found that the amino acid residue differences between enzyme from P. fluorescence and aeruginosa do marginally change some observed transient state kinetic parameters, even though the structure of the enzyme shows they have no direct role in catalysis. Thus, transient state kinetic analysis is an excellent tool to examine the role of amino acid residues in catalysis.
Entsch
B
B
Department of Biochemistry, Microbiology and Nutrition, University of New England, Armidale, N.S.W., Australia.
Ballou
D P
DP
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D009249:NADP; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; D010100:Oxygen