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Enzyme Nomenclature
Information on EC 1.14.99.67 - alpha-N-dichloroacetyl-p-aminophenylserinol N-oxygenase
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EC Tree
1.14.99.67 alpha-N-dichloroacetyl-p-aminophenylserinol N-oxygenaseIUBMB 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.
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The expected taxonomic range for this enzyme is: Streptomyces venezuelae
Reaction Schemes
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
cmlI
cmlI
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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
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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
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
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
?
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?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
?
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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
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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
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-
-
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
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-
-
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
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-
-
?
4-aminobenzoate + reduced phenazine methosulfate + 2 O2
4-nitrobenzoate + phenazine methosulfate + 2 H2O
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?
4-aminobenzoate + reduced phenazine methosulfate + 2 O2
4-nitrobenzoate + phenazine methosulfate + 2 H2O
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?
4-aminophenol + reduced phenazine methosulfate + 2 O2
4-nitrophenol + phenazine methosulfate + 2 H2O
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?
4-aminophenol + reduced phenazine methosulfate + 2 O2
4-nitrophenol + phenazine methosulfate + 2 H2O
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?
L-4-aminophenylalanine + reduced phenazine methosulfate + 2 O2
L-4-nitrophenylalanine + phenazine methosulfate + 2 H2O
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-
?
L-4-aminophenylalanine + reduced phenazine methosulfate + 2 O2
L-4-nitrophenylalanine + phenazine methosulfate + 2 H2O
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-
-
?
N-[(1R,2R)-1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
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-
-
?
N-[(1R,2R)-1-(4-aminophenyl)-1,3-dihydroxypropan-2-yl]-2,2-dichloroacetamide + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
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?
additional information
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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
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additional information
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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
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additional information
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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
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additional information
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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
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
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
?
-
-
-
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine ethosulfate + 2 O2
?
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-
-
?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
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?
(1R,2R)-(-)-2-(N-dichloroacetyl)-amino-1-(4-aminophenyl)-1,3-propanediol + reduced phenazine methosulfate + 2 O2
chloramphenicol + phenazine methosulfate + 2 H2O
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?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Iron
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
Iron
non-heme di-iron enzyme, iron content is 1.2-1.4 mol per mol of enzyme
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
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
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
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
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
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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
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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
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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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physiological function
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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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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
-
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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
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Release 2023.1 (January 2023)
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