NEW AEROBIC BENZOATE OXIDATION PATHWAY VIA BENZOYL-COENZYME-A AND 3-HYDROXYBENZOYL-COENZYME-A IN A DENITRIFYING PSEUDOMONAS SP

A denitrifying Pseudomonas sp. is able to oxidize aromatic compounds c ompletely to CO2, both aerobically and anaerobically. It is shown that benzoate is aerobically oxidized by a new degradation pathway via ben zoyl-coenzyme A (CoA) and 3-hydroxybenzoyl-CoA. The organism grew aero bically with benzoate, 3-hydroxybenzoate, and gentisate; catechol, 2-h ydroxybenzoate, and protocatechuate were not used, and 4-hydroxybenzoa te was a poor substrate. Mutants were obtained which were not able to utilize benzoate as the sole carbon source aerobically but still used 3-hydroxybenzoate or gentisate. Simultaneous adaptation experiments wi th whole cells seemingly suggested a sequential induction of enzymes o f a benzoate oxidation pathway via 3-hydroxybenzoate and gentisate. Ce lls grown aerobically with benzoate contained a benzoate-CoA ligase (A MP forming) (0.1 mumol min-1 mg-1) which converted benzoate but not 3- hydroxybenzoate into its CoA thioester. The enzyme of 130 kDa composed of two identical subunits of 56 kDa was purified and characterized. C ells grown aerobically with 3-hydroxybenzoate contained a similarly ac tive CoA ligase for 3-hydroxybenzoate, 3-hydroxybenzoate-CoA ligase (A MP forming). Extracts from cells grown aerobically with benzoate catal yzed a benzoyl-CoA- and flavin adenine dinucleotide-dependent oxidatio n of NADPH with a specific activity of at least 25 nmol NADPH oxidized min-1 mg of protein-1; NADH and benzoate were not used. This new enzy me, benzoyl-CoA 3-monooxygenase, was specifically induced during aerob ic growth with benzoate and converted [U-C-14]benzoyl-CoA stoichiometr ically to [C-14]3-hydroxybenzoyl-CoA.