A new anthranilic acid hydroxylase from Aspergillus niger. Purification and properties.

A new anthranilic acid hydroxylase which catalyzes the conversion of anthranilic acid to 2,3-dihydroxybenzoic acid, has been partially purified from crude extracts of the mycelial felts of Aspergillus niger, grown in the presence of anthranilic acid. Neither 3-hydroxyanthranilic acid nor salicylic acid is an intermediate in this conversion. The enzyme is stabilized by glutathione and requires substrate amounts of NADPH. The optimum pH for anthranilic acid hydroxylase activity is around 8.2. The Km values for anthranilic acid and NADPH at pH 8.2 and 30” are 0.15 and 0.16 mu, respectively. The partially purified enzyme is highly specific for anthranilic acid and does not act on related substances such as 3-hydroxyanthranilic acid, salicylic acid, m-hydroxybenzoic acid, methylanthranilic acid, and ethylanthranilic acid. Cupric, mercuric, and cadmium ions have a pronounced inhibitory effect on enzyme activity. Metal-chelating agents like o-phenanthroline, cr, cr’-dipyridyl, salicylaldoxime, and diethyl dithiocarbamate also inhibit the reaction. Sulfhydryl reagents such as p-hydroxymercuribenzoate and N-ethyhnaleimide irreversibly inhibit anthranilic acid hydroxylase activity. Crude extracts of the mycelium have the ability to hydroxylate anthranilic acid to 3-hydroxyanthranilic acid in addition to 2,3-dihydroxybenzoic acid. This activity, however, is lost on purification.