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Information on EC 1.14.99.68 - 4-aminobenzoate N-oxygenase
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EC Tree 1 Oxidoreductases
1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
1.14.99 Miscellaneous
1.14.99.68 4-aminobenzoate N-oxygenase
IUBMB Comments
The enzyme, characterized from the bacterium Streptomyces thioluteus, catalyses an early step in the biosynthesis of the antibiotic aureothin. It contains a carboxylate-bridged binuclear non-heme iron cluster. The native electron donor has not been identified, but is likely an iron-sulfur protein. The reaction mechanism involves formation of an extremely stable peroxo intermediate that catalyses three two-electron oxidations via a hydroxylamine and dihydroxylamine intermediates. cf. EC 1.14.99.67, N-[1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide N-oxygenase.
The expected taxonomic range for this enzyme is: Streptomyces thioluteus
Reaction Schemes
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Synonyms
4-aminobenzoate n-oxygenase, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
aurF
aurF
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-
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-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
4-aminobenzoate + reduced acceptor + 2 O2 = 4-nitrobenzoate + acceptor + 2 H2O
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PATHWAY SOURCE
PATHWAYS
MetaCyc
aureothin biosynthesis, spectinabilin biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
4-aminobenzoate,acceptor:oxygen oxidoreductase (N-hydroxylating)
The enzyme, characterized from the bacterium Streptomyces thioluteus, catalyses an early step in the biosynthesis of the antibiotic aureothin. It contains a carboxylate-bridged binuclear non-heme iron cluster. The native electron donor has not been identified, but is likely an iron-sulfur protein. The reaction mechanism involves formation of an extremely stable peroxo intermediate that catalyses three two-electron oxidations via a hydroxylamine and dihydroxylamine intermediates. cf. EC 1.14.99.67, N-[1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide N-oxygenase.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-aminobenzoate + reduced acceptor + 2 O2
4-nitrobenzoate + acceptor + 2 H2O
Substrates: -
Products: -
Products: -
?
additional information
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Substrates: the yield of the final product 4-nitrobenzoate is 37.2% when equimoles of reductants phenazine methosulfate and 4-aminobenzoic acid and 4 equimoles of ascorbate are used
Products: -
Products: -
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-aminobenzoate + reduced acceptor + 2 O2
4-nitrobenzoate + acceptor + 2 H2O
Substrates: -
Products: -
Products: -
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Iron
Manganese
crystal structure shows a binuclear manganese cluster
Iron
the iron-to-enzyme ratio is 2.2, protein shows a high selectivity for iron
Iron
the Fe2 II/II cluster in AurF reacts with O2 in the absence of substrate to form a stable (half-life of about 7 min at 20°C) adduct with spectroscopic properties characteristic of a peroxo-Fe2 III/III complex
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ascorbate
the in vitro enzymatic activity of AurF is best reconstituted in the presence of phenazine methosulfate and ascorbate
phenazine methosulfate
the in vitro enzymatic activity of AurF is best reconstituted in the presence of phenazine methosulfate and ascorbate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
physiological function
metabolism
comparison of AurF from Streptomyces thioluteus that catalyzes the formation of 4-nitrobenzoate from 4-aminobenzoate as a precursor to aureothin, whereas CmlI from Streptomyces venezuelae, cf. EC 1.14.99.68, catalyzes the ultimate aryl-amine to aryl-nitro step in chloramphenicol biosynthesis. The enzymes optimize efficiency by utilizing one of the reaction pathway intermediates as an in situ reductant for the diiron cluster, while simultaneously generating the next pathway intermediate
metabolism
the reaction starts with the binding of a dioxygen molecule to the diferrous center to generate a diferric peroxide complex, followed by the cleavage of the O-O bond, concertedly with the formation of the first N-O bond, which has a barrier of only 9.2 kcal/mol. Subsequently, the first-shell ligand Glu227 abstracts a proton from the substrate. After the delivery of two electrons from the external reductant and two protons from solution, a water molecule and the intermediate 4-hydroxylaminobenzoate are produced and the diferrous center is regenerated. The oxidation of the 4-hydroxylaminobenzoate intermediate requires the binding of a second dioxygen molecule to the diferrous center to generate the diferric peroxide complex. Similarly to the oxidation of 4-aminobenzoate, the O-O bond cleavage and the formation of the second N-O bond take place in a concerted step. The 4-nitrobenzoate product is formed after the release of two protons and two electrons from the substrate
physiological function
the key oxygenated intermediates in diiron arylamine oxygenases AurF and CmlI, EC 1.14.99.67, so-called P, are uniformly hydroperoxo species having similar structures rather than the believed peroxo species. A diferric-hydroperoxo P is proposed to be able to promote the arylamine N-oxygenation with highly accessible kinetics
physiological function
in-frame deletion of aurF yields a mutant, in which both N-oxidation activity and aureothin production is abolished. Aureothin biosynthesis in the mutant can be fully restored upon supplying the culture with synthetic 4-nitrobenzoate
physiological function
comparison with aminopyrrolnitrin oxygenase (PrnD). Hydroxylamine and nitroso intermediates are invoved in oxygenation reaction of arylamines
physiological function
AurF is a non-heme di-iron monooxygenase that catalyzes sequential oxidation of aminoarenes to nitroarenes via hydroxylamine and nitroso intermediates
physiological function
the Fe2 II/II cluster in AurF reacts with O2 in the absence of substrate to form a stable (half-life of about 7 min at 20°C) adduct with spectroscopic properties characteristic of a peroxo-Fe2 III/III complex. The intermediate complex decays rapidly (half-life of about 0.005 s at 20°C) when mixed with stoichiometric 4-aminobenzoate. Over 80% conversion of 4-aminobenzoate to 4-nitrobenzoate is observed upon addition of less than 0.3 equivalents of the amine substrate to a solution of the intermediate peroxo-Fe2 III/III complex. The complex might effect all three steps in the sequence
physiological function
peroxo-Fe2 III-III AurF oxidizes 4-hydroxylaminobenzoate Ar-NHOH. This reaction proceeds through to the Ar-NO2 final product, a four-electron oxidation, and produces Fe2 II-II AurF, with which O2 can combine to regenerate peroxo-Fe2 III-III AurF. Conversion of Ar-NHOH to Ar-NO2 requires only a single equivalent of O2 is fully catalytic in the absence of exogenous reducing equivalents
physiological function
reduced AurF's electronic and geometric structures are poised to react rapidly with O2. The active peroxo intermediate has a protonated peroxo bridge. The protonation activates peroxide for electrophilic/single-electron-transfer reactivity
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
structure at 2.1 A resolution shows a homodimer with a binuclear manganese cluster and an iron content of about 15%. An additional histidine residue in the coordination sphere causes the preference for manganese over iron. There is no oxo-bridge
structure of AurF in the oxidized state, and the cocrystal structure of AurF with its product 4-nitrobenzoate
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli as a fusion with an N-terminal maltose-binding protein
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Lee, J.; Zhao, H.
Mechanistic studies on the conversion of arylamines into arylnitro compounds by aminopyrrolnitrin oxygenase identification of intermediates and kinetic studies
Angew. Chem. Int. Ed. Engl.
45
622-625
2006
Streptomyces thioluteus (Q70KH9)
brenda
He, J.; Hertweck, C.
Biosynthetic origin of the rare nitroaryl moiety of the polyketide antibiotic aureothin Involvement of an unprecedented N-oxygenase
J. Am. Chem. Soc.
126
3694-3695
2004
Streptomyces thioluteus (Q70KH9)
brenda
Korboukh, V.; Li, N.; Barr, E.; Bollinger Jr., J.; Krebs, C.
A long-lived, substrate-hydroxylating peroxodiiron(III/III) intermediate in the amine oxygenase, AurF, from Streptomyces thioluteus
J. Am. Chem. Soc.
131
13608-13609
2009
Streptomyces thioluteus (Q70KH9)
brenda
Wang, C.; Chen, H.
Convergent theoretical prediction of reactive oxidant structures in diiron arylamine oxygenases AurF and CmlI peroxo or hydroperoxo?
J. Am. Chem. Soc.
139
13038-13046
2017
Streptomyces thioluteus (Q70KH9)
brenda
Park, K.; Li, N.; Kwak, Y.; Srnec, M.; Bell, C.B.; Liu, L.V.; Wong, S.D.; Yoda, Y.; Kitao, S.; Seto, M.; Hu, M.; Zhao, J.; Krebs, C.; Bollinger, J.M.; Solomon, E.I.
Peroxide activation for electrophilic reactivity by the binuclear non-heme iron enzyme AurF
J. Am. Chem. Soc.
139
7062-7070
2017
Streptomyces thioluteus (Q70KH9)
brenda
Zocher, G.; Winkler, R.; Hertweck, C.; Schulz, G.
Structure and action of the N-oxygenase AurF from Streptomyces thioluteus
J. Mol. Biol.
373
65-74
2007
Streptomyces thioluteus (Q70KH9)
brenda
Choi, Y.S.; Zhang, H.; Brunzelle, J.S.; Nair, S.K.; Zhao, H.
In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis
Proc. Natl. Acad. Sci. USA
105
6858-6863
2008
Streptomyces thioluteus (Q70KH9)
brenda
Li, N.; Korboukh, V.; Krebs, C.; Bollinger Jr., J.
Four-electron oxidation of p-hydroxylaminobenzoate to p-nitrobenzoate by a peroxodiferric complex in AurF from Streptomyces thioluteus
Proc. Natl. Acad. Sci. USA
107
15722-15727
2010
Streptomyces thioluteus (Q70KH9)
brenda
Wei, W.; Siegbahn, P.; Liao, R.
Mechanism of the dinuclear iron enzyme p-aminobenzoate N-oxygenase from density functional calculations
ChemCatChem
11
601-613
2019
Streptomyces thioluteus (Q70KH9)
-
brenda
Komor, A.; Jasniewski, A.; Que, L.; Lipscomb, J.
Diiron monooxygenases in natural product biosynthesis
Nat. Prod. Rep.
35
646-659
2018
Streptomyces thioluteus (Q70KH9)
brenda
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