A new variant of aerobic benzoate degradation has been found in a denitrifying bacterium in which benzoyl-CoA is the first intermediate [Altenschmidt, U., Oswald, B., Steiner, E., Herrmann, H. & Fuchs, G. (1993) New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp, J. Bacteriol. 175, 4851-4858)]. The initial reaction is catalyzed by benzoate-CoA ligase (AMP-forming), converting benzoate into benzoyl-CoA. The next step is 3-hydroxylation of benzoyl-CoA to 3-hydroxybenzoyl-CoA catalyzed by a flavin-nucleotide-dependent monooxygenase, benzoyl-CoA 3-monooxygenase. This novel enzyme has been purified and studied. It is specific for NADPH and requires the presence of a flavin nucleotide for activity; both FAD or FMN function similarly well as cofactor. Only benzoyl-CoA, but not benzoate, is hydroxylated. The protein is a monomer of M(r) 65,000 and is induced when cells are grown aerobically with benzoate. 3-Hydroxybenzoyl-CoA is further hydroxylated para to the hydroxyl group affording 2,5-dihydroxybenzoate (gentisate). This reaction requires another monooxygenase, 3-hydroxybenzoyl-CoA 6-monooxygenase, which is unspecific specific with respect to the pyridine nucleotide. Cells contain a second 6-monooxygenase activity which acts on free 3-hydroxybenzoate. Based on these and other data, the outlines of the new aerobic benzoate pathway have been deduced. The proposed intermediates are benzoyl-CoA, 3-hydroxybenzoyl-CoA, gentisate, maleylpyruvate, fumarylpyruvate and fumarate plus pyruvate.