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Information on EC 1.14.99.67 - alpha-N-dichloroacetyl-p-aminophenylserinol N-oxygenase
for references in articles please use BRENDA:EC1.14.99.67
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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
1.14.99.67 alpha-N-dichloroacetyl-p-aminophenylserinol N-oxygenase
IUBMB Comments
The enzyme, isolated from the bacterium Streptomyces venezuelae, is involved in the biosynthesis of the antibiotic chloramphenicol. It contains a carboxylate-bridged binuclear non-heme iron cluster. The components of the native electron chain have not been identified, although the immediate donor is likely to be an iron-sulfur protein. The reaction mechanism involves formation of an extremely stable peroxo intermediate that catalyses three individual two-electron oxidations via a hydroxylamine and a nitroso intermediates without releasing the intermediates. cf. EC 1.14.99.68, 4-aminobenzoate N-oxygenase.
The expected taxonomic range for this enzyme is: Streptomyces venezuelae
Reaction Schemes
showSynonyms
cmlI, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
cmlI
cmlI
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-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
alpha-N-dichloroacetyl-p-aminophenylserinol + reduced acceptor + 2 O2 = chloramphenicol + acceptor + 2 H2O
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PATHWAY SOURCE
PATHWAYS
MetaCyc
chloramphenicol biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
alpha-N-dichloroacetyl-p-aminophenylserinol,acceptor:oxygen oxidoreductase (N-hydroxylating)
The enzyme, isolated from the bacterium Streptomyces venezuelae, is involved in the biosynthesis of the antibiotic chloramphenicol. It contains a carboxylate-bridged binuclear non-heme iron cluster. The components of the native electron chain have not been identified, although the immediate donor is likely to be an iron-sulfur protein. The reaction mechanism involves formation of an extremely stable peroxo intermediate that catalyses three individual two-electron oxidations via a hydroxylamine and a nitroso intermediates without releasing the intermediates. cf. EC 1.14.99.68, 4-aminobenzoate 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
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
chloramphenicol + phenazine ethosulfate + 2 H2O
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
4-aminobenzoate + reduced phenazine methosulfate + 2 O2
4-nitrobenzoate + phenazine methosulfate + 2 H2O
4-aminophenol + reduced phenazine methosulfate + 2 O2
4-nitrophenol + phenazine methosulfate + 2 H2O
L-4-aminophenylalanine + reduced phenazine methosulfate + 2 O2
L-4-nitrophenylalanine + phenazine methosulfate + 2 H2O
N-[(1R,2R)-1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
?
Substrates: -
Products: -
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(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
?
Substrates: -
Products: -
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(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
chloramphenicol + phenazine ethosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
chloramphenicol + phenazine ethosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
4-aminobenzoate + reduced phenazine methosulfate + 2 O2
4-nitrobenzoate + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
4-aminobenzoate + reduced phenazine methosulfate + 2 O2
4-nitrobenzoate + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
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?
4-aminophenol + reduced phenazine methosulfate + 2 O2
4-nitrophenol + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
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4-aminophenol + reduced phenazine methosulfate + 2 O2
4-nitrophenol + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
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L-4-aminophenylalanine + reduced phenazine methosulfate + 2 O2
L-4-nitrophenylalanine + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
L-4-aminophenylalanine + reduced phenazine methosulfate + 2 O2
L-4-nitrophenylalanine + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
N-[(1R,2R)-1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
N-[(1R,2R)-1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
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additional information
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Substrates: no acceptor: flavin mononucleotide. The reaction shows a biphasic behavior, with a fast step consuming significant amounts of substrate within 15 s, and a slower step afterward
Products: -
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additional information
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Substrates: CmlI peroxo reacts with a range of arylamine substrates by an apparent second-order process. Chloramphenicol is efficiently produced from the free arylamine precursor. No substrate: L-4-aminophenylalanine
Products: -
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additional information
?
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Substrates: no acceptor: flavin mononucleotide. The reaction shows a biphasic behavior, with a fast step consuming significant amounts of substrate within 15 s, and a slower step afterward
Products: -
Products: -
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additional information
?
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Substrates: CmlI peroxo reacts with a range of arylamine substrates by an apparent second-order process. Chloramphenicol is efficiently produced from the free arylamine precursor. No substrate: L-4-aminophenylalanine
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
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
?
Substrates: -
Products: -
Products: -
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
?
Substrates: -
Products: -
Products: -
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
Products: -
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
Substrates: -
Products: -
Products: -
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Iron
Iron
non-heme di-iron enzyme, iron content is 1.2-1.4 mol per mol of enzyme
Iron
enzyme contains a nonheme dinuclear iron cluster, with 2.1 iron atoms per monomer. Addition of O2 to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t1/2 is 3 h at 4°C) that is assigned as a peroxodiferric species. CmlI-peroxo does not have a my-eta1:eta1-peroxo ligand, a my-eta1:eta2-peroxo ligand is proposed
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
brenda
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, cf. EC 1.14.99.68, that catalyzes the formation of 4-nitrobenzoate from 4-aminobenzoate as a precursor to aureothin, whereas CmlI from Streptomyces venezuelae 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. For CmlI, this reduction allows mid-pathway regeneration of the peroxo intermediate required to complete the biosynthesis
metabolism
-
comparison of AurF from Streptomyces thioluteus, cf. EC 1.14.99.68, that catalyzes the formation of 4-nitrobenzoate from 4-aminobenzoate as a precursor to aureothin, whereas CmlI from Streptomyces venezuelae 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. For CmlI, this reduction allows mid-pathway regeneration of the peroxo intermediate required to complete the biosynthesis
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physiological function
CmlI is a non-heme di-iron enzyme catalyzing N-oxygenation of an amine group, one of the steps in the biosynthesis of the antibiotic chloramphenicol
physiological function
during the catalytic cycle substrate N-[(1R,2R)-1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide reacts with a long-lived peroxo intermediate to form a NH(OH)-chloramphenicol species and diferric CmlI. Then the NH(OH)-chloramphenicol re-reduces the enzyme diiron cluster, allowing the peroxo species to re-form upon O2 binding, while itself being oxidized to NO-chloramphenicol. Finally, the re-formed peroxo species oxidizes NO-chloramphenicol to chloramphenicol with incorporation of a second O2-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and may occur in one active site
physiological function
diferrous CmlI can react with NH2-chloramphenicol species and O2 in either order to form a peroxo-NH2-chloramphenicol intermediate. Peroxo-NH2-chloramphenicol undergoes rapid oxygen transfer to form a diferric CmlI complex with the aryl-hydroxylamine [NH(OH)-chloramphenicol] pathway intermediate. Diferric CmlI-NH(OH)-chloramphenicol undergoes a rapid internal redox reaction to form a differous CmlI-nitroso-chloramphenicol complex. O2 binding results in formation of peroxo-NO-chloramphenicol that converts to differic CmlI-chloramphenicol by enzyme-mediated oxygen atom transfer. There is little dissociation of pathway intermediates as the reaction progresses
physiological function
the key oxygenated intermediates in diiron arylamine oxygenases AurF, EC 1.14.99.68, and CmlI, 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
-
CmlI is a non-heme di-iron enzyme catalyzing N-oxygenation of an amine group, one of the steps in the biosynthesis of the antibiotic chloramphenicol
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physiological function
-
during the catalytic cycle substrate N-[(1R,2R)-1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide reacts with a long-lived peroxo intermediate to form a NH(OH)-chloramphenicol species and diferric CmlI. Then the NH(OH)-chloramphenicol re-reduces the enzyme diiron cluster, allowing the peroxo species to re-form upon O2 binding, while itself being oxidized to NO-chloramphenicol. Finally, the re-formed peroxo species oxidizes NO-chloramphenicol to chloramphenicol with incorporation of a second O2-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and may occur in one active site
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physiological function
-
diferrous CmlI can react with NH2-chloramphenicol species and O2 in either order to form a peroxo-NH2-chloramphenicol intermediate. Peroxo-NH2-chloramphenicol undergoes rapid oxygen transfer to form a diferric CmlI complex with the aryl-hydroxylamine [NH(OH)-chloramphenicol] pathway intermediate. Diferric CmlI-NH(OH)-chloramphenicol undergoes a rapid internal redox reaction to form a differous CmlI-nitroso-chloramphenicol complex. O2 binding results in formation of peroxo-NO-chloramphenicol that converts to differic CmlI-chloramphenicol by enzyme-mediated oxygen atom transfer. There is little dissociation of pathway intermediates as the reaction progresses
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physiological function
-
the key oxygenated intermediates in diiron arylamine oxygenases AurF, EC 1.14.99.68, and CmlI, 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
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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). CMLI_STRVP
Streptomyces venezuelae (strain ATCC 10712 / CBS 650.69 / DSM 40230 / JCM 4526 / NBRC 13096 / PD 04745)
339
0
38249
Swiss-Prot
Secretory Pathway (Reliability: 4)
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of the fully active, N-terminally truncated CmlIDelta33 in the chemically reduced Fe2+/Fe2+ state and a cis my-1,2(eta1:eta1)-peroxo complex. CmlIDelta33 crystallized at pH 6.8 lacks the oxo-bridge apparent from the enzyme optical spectrum in solution at higher pH. In its place, residue E236 forms a my-1,3(eta1:eta2) bridge between the irons in both models. This orientation of E236 stabilizes a helical region near the cluster which closes the active site to substrate binding in contrast to the open site found for homologous arylamine oxygenase AurF
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Komor, A.; Rivard, B.; Fan, R.; Guo, Y.; Que, L.; Lipscomb, J.
CmlI N-oxygenase catalyzes the final three steps in chloramphenicol biosynthesis without dissociation of intermediates
Biochemistry
56
4940-4950
2017
Streptomyces venezuelae (F2RB83), Streptomyces venezuelae DSM 40230 (F2RB83)
brenda
Makris, T.; Vu, V.; Meier, K.; Komor, A.; Rivard, B.; Muenck, E.; Que, L.; Lipscomb, J.
An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway
J. Am. Chem. Soc.
137
1608-1617
2015
Streptomyces venezuelae (F2RB83), Streptomyces venezuelae DSM 40230 (F2RB83)
brenda
Komor, A.; Rivard, B.; Fan, R.; Guo, Y.; Que, L.; Lipscomb, J.
Mechanism for six-electron aryl-N-oxygenation by the non-heme diiron enzyme CmlI
J. Am. Chem. Soc.
138
7411-7421
2016
Streptomyces venezuelae (F2RB83), Streptomyces venezuelae DSM 40230 (F2RB83)
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 venezuelae (F2RB83), Streptomyces venezuelae DSM 40230 (F2RB83)
brenda
Knoot, C.; Kovaleva, E.; Lipscomb, J.
Crystal structure of CmlI, the arylamine oxygenase from the chloramphenicol biosynthetic pathway
J. Biol. Inorg. Chem.
21
589-603
2016
Streptomyces venezuelae (F2RB83), Streptomyces venezuelae DSM 40230 (F2RB83)
brenda
Lu, H.; Chanco, E.; Zhao, H.
CmlI is an N-oxygenase in the biosynthesis of chloramphenicol
Tetrahedron
68
7651-7654
2012
Streptomyces venezuelae (F2RB83), Streptomyces venezuelae DSM 40230 (F2RB83)
brenda
Komor, A.; Jasniewski, A.; Que, L.; Lipscomb, J.
Diiron monooxygenases in natural product biosynthesis
Nat. Prod. Rep.
35
646-659
2018
Streptomyces venezuelae (F2RB83), Streptomyces venezuelae DSM 40230 (F2RB83)
brenda
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