Reactions of 1-deaza-FAD-substituted phenol hydroxylase and melilotate hydroxylase.
The flavin prosthetic group (FAD) of the aromatic hydroxylases melilotate hydroxylase (EC 1.14.13.4) and phenol hydroxylase (EC 1.14.13.7) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). Neither modified enzyme could hydroxylate its substrate, both catalyzed the oxidation of NAD(P)H to NAD(P)+ and H2O2. The rate of the reduction of the enzymes by NAD(P)H was increased by the binding of substrate. Both enzymes formed a detectable flavin C(4a) hydroperoxide intermediate upon reaction of the reduced enzyme-substrate complex with oxygen. Reduced 1-deaza-FAD phenol hydroxylase also showed a detectable C(4a) hydroperoxide intermediate when reacted with oxygen in the absence of substrate. The C(4a) hydroperoxide of 1-deaza-FAD phenol hydroxylase, in the absence of phenol, decayed to an intermediate which showed a perturbed oxidized enzyme spectrum, Eox. This intermediate in turn decayed to give the original oxidized enzyme. In the presence of phenol, a second oxidized species with a perturbed spectrum, intermediate X, was apparent after formation of the flavin C(4a) hydroperoxide and before Eox formation. Steady state kinetic analysis of 1-deaza-FAD phenol hydroxylase demonstrated that the Eox to Eox conversion was not in the catalytic cycle. During turnover Eox was reduced by NADPH.
D001051:Apoenzymes / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction
D001051:Apoenzymes; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C023406:1-deaza-FAD; D006899:Mixed Function Oxygenases; C021549:melilotate 3-monooxygenase; C022336:phenol 2-monooxygenase