NIH shift in flavin-dependent monooxygenation: Mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase

The flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase from the eubacterium Azoarcus evansii catalyzes the dearomatization of 2-aminobenzoyl-CoA. The reaction consists in an O(2)-dependent monooxygenation at the benzene position 5, which is followed immediately by an NADH-dependent hydrogenation of the intermediate at the same catalytic locus. The reaction was studied by (1)H, (2)H, and (13)C NMR spectroscopy of the products. The main product was characterized as 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA by two-dimensional NMR spectroscopy. Thus, [5-(2)H]2-aminobenzoyl-CoA was converted into [6-(2)H]5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA, indicating a 5 → 6 shift of the [5-(2)H] label. Label from NAD(2)H was transferred to the 3 position of the cyclic eneamine, whereas label from solvent D(2)O was incorporated into the 4 and the 6 positions of 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA. The labeling pattern is compatible with the monooxygenation proceeding via what is formally an NIH shift, yielding 5-oxo-2-aminocyclohex-1,3-diene-1-carboxyl-CoA as a protein-bound intermediate. It is suggested that this shift in flavin-dependent monooxygenation may have general validity.