The Purification and Properties of 4-Hydroxyisophthalate Hydroxylase from Pseudomonas putida NCIB 9866.

4-Hydroxyisophthalate hydroxylase has been purified from cells grown with 2,4-xylenol and gave a single protein band on polyacrylamide-gel electrophoresis after a 24-fold purification. In the reaction, 4-hydroxyisophthalate was converted to protocatechuate by an oxidative decarboxylation and the reaction stoichiometry is typical of a monooxygenase with an external electron donor. The enzyme can function with either NADH (Km 105 μM) or NADPH (Km 71 μM). Besides 4-hydroxyisophthalate (Km 42 μM), 5-sulphosalicylate was the only aromatic substrate found for the enzyme. The enzyme has a molecular weight of about 103000, consisting of two identical polypeptide chains, and contains one mole of FAD per mole of protein. The FAD could not be replaced by FMN m reconstituting active enzyme from native apoenzyme. The formation of an enzyme-substrate complex was detected by the change in flavin spectrum when 4-hydroxyisophthalate was added to the enzyme. Rapid reduction of the enzyme-bound FAD by NADPH only occurred in the presence of substrate. No stable flavin semiquinones were observed in the titration with NADPH or during anaerobic photoreduction in the presence of EDTA. Although a number of substrate analogues inhibit the enzyme no non-hydroxylatable effectors of the NADPH oxidation were found. The enzyme was inhibited by sulphydryl-binding reagents and not protected by substrate. Addition of sulphydryl compounds was essential for stabilization of the enzyme during purification. Extracts of cells grown with 2,4-xylenol and 4-hydroxyisophthalate also contain a separate 4-hydroxybenzoate hydroxylase. [ABSTRACT FROM AUTHOR]