Characterization and Evolution of Anthranilate 1,2-Dioxygenase from Acinetobacter sp. Strain ADP1

摘要: The two-component anthranilate 1,2-dioxygenase of the bacterium Acinetobacter sp. strain ADP1 was expressed in Escherichia coli and purified to homogeneity. This enzyme converts (2-aminobenzoate) catechol with insertion both atoms O(2) consumption one NADH. terminal oxygenase component formed an alpha(3)beta(3) hexamer 54- 19-kDa subunits. Biochemical analyses demonstrated Rieske-type [2Fe-2S] center mononuclear nonheme iron each large subunit. reductase component, which transfers electrons from NADH found contain approximately flavin adenine dinucleotide ferredoxin-type per 39-kDa monomer. Activities combined components were measured as rates quantities oxidation, substrate disappearance, product appearance, consumption. Anthranilate conversion stoichiometrically coupled oxidation analog benzoate converted a nonaromatic 1,2-diol similarly tight coupling. latter activity is identical that related 1, 2-dioxygenase. A variant 1,2-dioxygenase, previously convey temperature sensitivity vivo because methionine-to-lysine change subunit, characterized. M43K variant, however, did not hydroxylate or at either permissive (23 degrees C) nonpermissive (39 growth temperatures. wild-type efficiently methylated halogenated benzoates, despite its sequence similarity broad-substrate specific dioxygenases do. Phylogenetic trees alpha beta subunits these act on natural xenobiotic substrates indicated evolved common ancestral two-subunit component.