2-Aminobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme : studies on the stoichiometry and the course of the reaction

Abstract

The reaction catalyzed by 2-aminobenzoyl-coenzyme-A monooxygenase/reductase from a denitrifying Pseudomonas sp. has been investigated. 2-Aminobenzoyl-CoA and 2-amino[carboxy-14C]benzoyl-CoA were synthesized enzymatically using 2-aminobenzoyl-CoA synthetase from the same organism. The product was purified by chromatography and characterized by ultraviolet/visible and 1H-NMR spectroscopy. The conversion of 2-aminobenzoyl-CoA catalyzed by the monooxygenase/reductase requires NADH and oxygen, and yields at least two different products depending on the relative concentration of NADH. At [NADH]< Km (40 μM), i.e. [NADH]/[2-aminobenzoyl-CoA] ~ 0.02 0.05, the main product is probably a hydroxylated derivative of 2-aminobenzoyl-CoA, which is characterized by an absorbance maximum around 375 nm. When [NADH]/[2-aminobenzoyl-CoA] ~ 2 5, the predominant product is a non-aromatic coenzyme A thioester (λmax ~ 320 nm). The stoichiometry in this case is 2.1 2.4 mol NADH oxidized (mol oxygen consumed)−1 (mol 2-aminobenzoyl-CoA metabolized)−1. The product is extremely unstable in the acidic pH range and undergoes decarboxylation in a few minutes at pH < 5. Some degree of stabilisation is obtained upon reduction with sodium borohydride, probably resulting in a further reduced non-aromatic coenzyme-A thioester.