Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD.
The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP; D010084:Oxidation-Reduction; D011485:Protein Binding; D011551:Pseudomonas fluorescens / Q000201:enzymology; D011809:Quinones; D013053:Spectrophotometry; D013379:Substrate Specificity
D011809:Quinones; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C023406:1-deaza-FAD; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase