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 O2-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 1H, 2H, and 13C 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, [5-2H]2-aminobenzoyl-CoA was converted into [6-2H]5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA, indicating a 5 --> 6 shift of the [5-2H] label. Label from NAD2H was transferred to the 3 position of the cyclic eneamine, whereas label from solvent D2O 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.
Fakultät Biologie, Universität Konstanz, P.O. Box 5560-M644, D-78457 Konstanz, Germany.
S
S
Hartmann
C
C
Hultschig
W
W
Eisenreich
G
G
Fuchs
A
A
Bacher
S
S
Ghisla
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes; D003903:Deuterium; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D011549:Pseudomonas / Q000201:enzymology; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism
D000214:Acyl Coenzyme A; D002247:Carbon Isotopes; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D003903:Deuterium; D006899:Mixed Function Oxygenases; C062023:2-aminobenzoyl-CoA monooxygenase-reductase