Studies on p-Hydroxybenzoate Hydroxylase from Pseudomonas putida

• Published in: The Journal of biological chemistry Vol. 244; no. 20; pp. 5644 - 5655
• Main Authors: Hesp, Barrie, Calvin, Melvin, Hosokawa, Keiichi
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25.10.1969
• Subjects: Benzoates; Binding Sites; Carbon Isotopes; Chemical Phenomena; Chemistry; Chromatography; Chromatography, DEAE-Cellulose; Chromatography, Gel; Electron Transport; Enzyme Induction; Flavin-Adenine Dinucleotide; Helium; Hydrogen-Ion Concentration; Kinetics; Methods; Mixed Function Oxygenases - biosynthesis; Mixed Function Oxygenases - isolation & purification; Models, Biological; Models, Chemical; Molecular Weight; NADP; Phenols; Pseudomonas - enzymology; Spectrophotometry; Spectrum Analysis; Ultracentrifugation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)63609-9

The mechanism of enzymatic hydroxylation was studied by the use of p -hydroxybenzoate hydroxylase. The enzyme preparation was obtained in crystalline form from Pseudomonas putida , M-6, which was grown on p -hydroxybenzoate solely as an inducer-substrate. The enzyme was homogeneous upon sedimentation analysis. The molecular weight was 93,622. The enzyme resembled the analogous enzyme from P. putida , strain A-3.12, in all other respects examined. The activity was maximum at pH 8.25 in tris(hydroxymethyl)-aminomethane-HCl buffer and at pH 7.0 in potassium phosphate buffer. One molecule of FAD is tightly bound per enzyme molecule. Neither flavin mononucleotide nor riboflavin was effective in restoring the activity to the apoenzyme prepared by acid treatment of the holoenzyme. Only FAD could do this. Substrate specificity was so strict that even close analogues such as benzoate, phenol, salicylate, p -hydroxybenzoic acid methyl ester, and the methoxy derivative of p -hydroxybenzoate were inactive in the stimulation of enzyme-catalyzed oxidation of NADPH, a specific electron donor. In anaerobic conditions, produced by bubbling helium gas through the system, FAD bound to p -hydroxybenzoate hydroxylase was reduced stoichiometrically by NADPH in the presence of p -hydroxybenzoate. On introduction of air, the reduced enzyme was quickly reoxidized. With 14 C-labeled p -hydroxybenzoate, it was demonstrated that reduced p -hydroxybenzoate hydroxylase was fully active in converting p -hydroxybenzoate to protocatechuate during aerobic oxidation of reduced enzyme. The production of protocatechuate was less than 0.6 to 0.7 of the amount of reduced enzyme. This may be due to partial inactivation of the enzyme during deoxygenation of the system, and uncoupling of electron transfer by a possible occurrence of substrate-free reduced enzyme, as demonstrated by chemical reduction. Spectrophotometric study showed that the holoenzyme was reduced stoichiometrically with dithionite in either the presence or absence of p -hydroxybenzoate. There was no change in the spectrum, which could be considered characteristic of flavin semiquinone during dithionite titration of substrate-free enzyme under anaerobic conditions. The circular dichroism (CD) spectrum of the holoenzyme in the absence of p -hydroxybenzoate had a strong positive band at 367 nm and a strong negative one at 448 nm. Since free FAD has a positive band at 340 nm and a negative one at 375 nm, FAD bound to the holoenzyme must have been subject to modification either in conformation or in chemical nature. By addition of p -hydroxybenzoate the positive band in the CD spectrum of substrate-free enzyme shifted to longer wave lengths and the negative one broadened generally. The visible region spectrum (350 to 500 nm) of the enzyme was slightly but significantly changed by addition of p -hydroxybenzoate, but the changes in the CD spectrum were remarkable. Addition of benzoate to substrate-free holoenzyme caused CD spectral changes similar to those caused by p -hydroxybenzoate. Phenol was ineffective in causing significant change in the CD spectrum. These facts indicate that the carboxyl, but not the hydroxyl, moiety of p -hydroxybenzoate is directly involved in binding the substrate to the enzyme in association with FAD. Other analogues were all inactive in giving rise to a significant change in the CD spectrum, indicating that enzyme-substrate binding is highly specific.