Purification and properties of 4-aminobenzoate hydroxylase, a new monooxygenase from Agaricus bisporus.
A new FAD-dependent monooxygenase, 4-aminobenzoate hydroxylase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate and forms 4-hydroxyaniline in the presence of NAD(P)H and O2 has been purified to homogeneity by ammonium sulfate fractionation, affinity chromatography, chromatofocusing, and Sephadex G-100 chromatography from Agaricus bisporus, a common edible mushroom. The molecular weight of the enzyme, which consists of a single polypeptide, is 49,000. The enzyme contains 0.91 mol of FAD/mol of enzyme. Stoichiometric studies show that 1 mol of 4-aminobenzoate is converted to an equimolecular amount of 4-hydroxyaniline and CO2 with the consumption of 1 mol each of NADH and molecular oxygen. Results obtained isotopically with 18O2 show that one atom of molecular oxygen is incorporated into 4-hydroxyaniline formed from 4-aminobenzoate. The enzyme is most active between pH 6.5 and 8.0 in the oxidation of NADH and between pH 6.0 and 7.5 in the case of NADPH. The Km values for 4-aminobenzoate, NADH, and O2 are 20.4, 13.6, and 200 microM, respectively, and that for NADPH is 133 microM. Other substituted benzoates with free amino and carboxyl groups in the ortho or para position (e.g. 4-aminosalicylate and anthranilate) serve as substrates for hydroxylation, but, in these cases, H2O2 is formed simultaneously with the hydroxylation. The enzyme is insensitive to the chelators of iron and copper, sodium arsenite, and KCN. Heavy metal ions and p-chloromercuribenzoate severely inhibit the enzyme enzyme
D010129:4-Aminobenzoic Acid / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000838:Anions / Q000494:pharmacology; D001487:Basidiomycota / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D000838:Anions; D004791:Enzyme Inhibitors; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D006899:Mixed Function Oxygenases; C050092:4-aminobenzoate hydroxylase; D010129:4-Aminobenzoic Acid