Reactions of 1-deaza-FAD-substituted phenol hydroxylase and melilotate hydroxylase

K. Detmer, Lawrence M Schopfer, V. Massey

Research output: Contribution to journalArticle

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Abstract

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 (carmon 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, E*(ox). 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 E*(ox) formation. Steady state kinetic analysis of 1-deaza-FAD phenol hydroxylase demonstrated that the E*(ox) to E(ox) conversion was not in the catalytic cycle. During turnover E*(ox) was reduced by NADPH.

Original languageEnglish (US)
Pages (from-to)1532-1538
Number of pages7
JournalJournal of Biological Chemistry
Volume259
Issue number3
StatePublished - Jan 1 1984

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melilotate 3-monooxygenase
phenol 2-monooxygenase
Hydrogen Peroxide
Enzymes
NAD
Substrates
Phenol
Oxygen
Reaction intermediates
Flavin-Adenine Dinucleotide
Mixed Function Oxygenases
Prosthetics
NADP
1-deaza-FAD
Nitrogen

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reactions of 1-deaza-FAD-substituted phenol hydroxylase and melilotate hydroxylase. / Detmer, K.; Schopfer, Lawrence M; Massey, V.

In: Journal of Biological Chemistry, Vol. 259, No. 3, 01.01.1984, p. 1532-1538.

Research output: Contribution to journalArticle

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abstract = "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 (carmon 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, E*(ox). 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 E*(ox) formation. Steady state kinetic analysis of 1-deaza-FAD phenol hydroxylase demonstrated that the E*(ox) to E(ox) conversion was not in the catalytic cycle. During turnover E*(ox) was reduced by NADPH.",
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N2 - 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 (carmon 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, E*(ox). 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 E*(ox) formation. Steady state kinetic analysis of 1-deaza-FAD phenol hydroxylase demonstrated that the E*(ox) to E(ox) conversion was not in the catalytic cycle. During turnover E*(ox) was reduced by NADPH.

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