Information on EC 1.14.12.22 - carbazole 1,9a-dioxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.12.22
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RECOMMENDED NAME
GeneOntology No.
carbazole 1,9a-dioxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
9H-carbazole + NAD(P)H + H+ + O2 = 2'-aminobiphenyl-2,3-diol + NAD(P)+
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
carbazole degradation
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Dioxin degradation
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Metabolic pathways
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
9H-carbazole,NAD(P)H:oxygen oxidoreductase (2,3-hydroxylating)
This enzyme catalyses the first reaction in the pathway of carbazole degradation. The enzyme attacks at the 1 and 9a positions of carbazole, resulting in the formation of a highly unstable hemiaminal intermediate that undergoes a spontaneous cleavage and rearomatization, resulting in 2'-aminobiphenyl-2,3-diol. In most bacteria the enzyme is a complex composed of a terminal oxygenase, a ferredoxin, and a ferredoxin reductase. The terminal oxygenase component contains a nonheme iron centre and a Rieske [2Fe-2S] iron-sulfur cluster.
CAS REGISTRY NUMBER
COMMENTARY hide
194812-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain KA1
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
9-fluorenone + NAD(P)H + H+ + O2
1,1a-dihydroxy-1-hydrofluoren-9-one + NAD(P)+
show the reaction diagram
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angular dioxygenation, yield 8-12%
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?
anthracene + NAD(P)H + H+ + O2
cis-1,2-dihydroxy-1,2-dihydroanthracene + NAD(P)+
show the reaction diagram
biphenyl + NAD(P)H + H+ + O2
2-hydroxybiphenyl + 3-hydroxybiphenyl + biphenyl dihydrodiol + NAD(P)+
show the reaction diagram
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in Escherichia coli expressing terminal oxygenase gene CarAa and ferredoxin component gene CarAc, ratio of products depends on reaction time. Synthesis of up to 46% biphenyl dihydrodiol
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?
biphenyl + NAD(P)H + H+ + O2
cis-2,3-dihydroxy-2,3-dihydrobiphenyl + 2-hydroxybiphenyl + 3-hydroxybiphenyl + NAD(P)+
show the reaction diagram
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lateral dioxygenation to cis-2,3-dihydroxy-2,3-dihydrobiphenyl, yield 85-90%, and to dihydrodiol, yield 9-11%
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?
biphenyl + NAD(P)H + H+ + O2
cis-2,3-dihydroxy-2,3-dihydrobiphenyl + NAD(P)+
show the reaction diagram
carbazole + NAD(P)H + H+ + O2
2'-aminobiphenyl-2,3-diol + NAD(P)+
show the reaction diagram
carbazole + NADH + H+ + O2
2'-aminobiphenyl-2,3-diol + NAD+
show the reaction diagram
carbazole + NADPH + H+ + O2
2'-aminobiphenyl-2,3-diol + NADP+
show the reaction diagram
dibenzo-p-dioxin + NAD(P)H + H+ + O2
2,2',3-trihydroxybiphenyl ether + NAD(P)+
show the reaction diagram
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26% 2,2',3-trihydroxybiphenyl ether in Escherichia coli expressing terminal oxygenase gene CarAa and ferredoxin component gene CarAc
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?
dibenzo-p-dioxin + NAD(P)H + H+ + O2
2,2',3-trihydroxydiphenyl ether + NAD(P)+
show the reaction diagram
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?
dibenzo-p-dioxin + NAD(P)H + H+ + O2
? + NAD(P)+
show the reaction diagram
dibenzofuran + NAD(P)H
? + NAD(P)+
show the reaction diagram
dibenzofuran + NAD(P)H + H+ + O2
2,2',3-trihydroxybiphenyl + NAD(P)+
show the reaction diagram
dibenzothiophene + NAD(P)H + H+ + O2
dibenzothiophene-5-oxide + NAD(P)+
show the reaction diagram
dibenzothiophene + NADH + H+ + O2
dibenzothiophene sulfoxide + NAD+ + H2O
show the reaction diagram
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monooxygenation, yield 99-100%
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?
fluoranthene + NAD(P)H + H+ + O2
cis-2,3-dihydroxy-2,3-dihydrofluoranthene + monohydroxyfluoranthene + NAD(P)+
show the reaction diagram
fluoranthene + NAD(P)H + H+ + O2
cis-2,3-dihydroxy-2,3-dihydrofluoranthene + NAD(P)+
show the reaction diagram
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poor substrate
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?
fluorene + NAD(P)H + H+ + O2
9-fluorenol + ? + NAD(P)+
show the reaction diagram
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monooxygenation to 9-fluorenol, yield 3-5%, and lateral dioxygenation to dihydrodiol, yield 5-8%
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?
fluorene + NAD(P)H + H+ + O2
9-fluorenone + 9-fluorenol + monohydroxyfluorene + fluorene dihydrodiol + NAD(P)+
show the reaction diagram
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in Escherichia coli expressing terminal oxygenase gene CarAa and ferredoxin component gene CarAc, ratio of products depends on reaction time
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?
fluorene + NAD(P)H + H+ + O2
9-hydroxyfluorene + NAD(P)+
show the reaction diagram
N-ethylcarbazole + NAD(P)H + H+ + O2
? + NAD(P)+
show the reaction diagram
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-
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?
N-methylcarbazole + NAD(P)H + H+ + O2
? + NAD(P)+
show the reaction diagram
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?
naphthalene + NAD(P)H + H+ + O2
1-naphthol + cis-1,2-dihydroxy-1,2-dihydronaphthalene + NAD(P)+
show the reaction diagram
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7.3% 1-naphthol and 93% cis-1,2-dihydroxy-1,2-dihydronaphthalene in Escherichia coli expressing terminal oxygenase gene CarAa and ferredoxin component gene CarAc
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?
naphthalene + NAD(P)H + H+ + O2
cis-1,2-dihydroxy-1,2-dihydronaphthalene + 1-naphthol + NAD(P)+
show the reaction diagram
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lateral dioxygenation, yield 65-70%
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?
naphthalene + NAD(P)H + H+ + O2
cis-1,2-dihydroxy-1,2-dihydronaphthalene + NAD(P)+
show the reaction diagram
phenanthrene + NAD(P)H + H+ + O2
? + NAD(P)+
show the reaction diagram
phenanthrene + NAD(P)H + H+ + O2
phenanthrene dihydrodiol + monohydroxyphenanthrene + NAD(P)+
show the reaction diagram
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70% phenanthrene dihydrodiol and 9% monohydroxyphenanthrene in Escherichia coli expressing terminal oxygenase gene CarAa and ferredoxin component gene CarAc
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?
phenazine + NAD(P)H + H+ + O2
? + NAD(P)+
show the reaction diagram
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?
phenothiazine + NAD(P)H + H+ + O2
? + NAD(P)+
show the reaction diagram
18 h reaction time, 7.6% substrate remaining
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?
phenoxathiin + NAD(P)H + H+ + O2
2,2',3-trihydroxydiphenyl sulfide + NAD(P)+
show the reaction diagram
phenoxazine + NAD(P)H + H+ + O2
? + NAD(P)+
show the reaction diagram
18 h reaction time, 1.1% substrate remaining
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?
xanthene + NAD(P)H + H+ + O2
2,2',3-trihydroxydiphenylmethane + NAD(P)+
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
1 mol of His-tagged CarAd contains 1 mol of FAD
Ferredoxin
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NAD(P)H
NADH
both NADH and NADPH are effective as electron donors for His-tagged ferrdoxin reductase CarAd. The ratio kcat/Km for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay
NADPH
both NADH and NADPH are effective as electron donors for His-tagged ferrdoxin reductase CarAd. The ratio kcat/Km for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0034
NADH
CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C
0.182
NADPH
CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2900
NADH
Pseudomonas resinovorans
Q8GI14, Q8GI16
CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C
8
130
NADPH
Pseudomonas resinovorans
Q8GI14, Q8GI16
CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C
5
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8.5
maximum cytochrome c reductase activity of subunit CarAd
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 45
maximum cytochrome c reductase activity of subunit CarAd
PDB
SCOP
CATH
ORGANISM
UNIPROT
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
Janthinobacterium sp. (strain J3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12300
x * 12300, calculated, x * 13000, SDS-PAGE, for ferredoxin component CARDO-F
36000
x * 36000, SDS-PAGE of ferredoxin reductuase subunit
38800
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gel filtration
43000
x * 43000, SDS-PAGE and calculated, subunit CarAa
43782
3 * 44000, SDS-PAGE, 3 * 43782, calculated, subunit CarAa
43900
3 * 43900, calculated, and 45000, SDS-PAGE of terminal oxygenase component
44000
3 * 44000, SDS-PAGE, 3 * 43782, calculated, subunit CarAa
124000
gel filtration of terminal oxygenase component
132000
gel filtration, subunit CarAa
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
trimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structures of the reduced carbazole-bound, dioxygen-bound, and both carbazole- and dioxygen-bound subunits CARDO-O:CARDO-F binary complex structures at 1.95, 1.85, and 2.00 A resolution, using the catalytic terminal oxygenase subunit from Janthinobacterium sp. J3 and ferredoxin from Pseudomonas resinovorans CA10. Catalytic mechanism is as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O–O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron
comparison of crystal structures of the oxygenase and ferredoxin components to the CARDOs from Pseudomonas resinovorans CA10, Janthinobacterium sp. J3, Novosphingobium sp. KA1, and Nocardioides aromaticivorans IC177 which are grouped into classes III, III, IIA, and IIB, respectively. The comparison suggests residues in common between class IIB and class III CARDOs that are important for interactions between ferredoxin and oxygenase. In the class IIB CARDOs, these include His75 and Glu71 in ferredoxin and Lys20 and Glu357 in the oxygenase for electrostatic interactions, and Phe74 and Pro90 in ferredoxin and Trp21, Leu359, and Val367 in the oxygenase for hydrophobic interactions
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crystal structure of oxygenase component CARDO-O at a resolution of 1.95 A, and of selenomethione derivative to 2.3 A resolution. The alpha3 trimeric overall structure of the CARDO-O molecule roughly corresponds to the alpha3 partial structures of other terminal oxygenase components of Rieske non-heme iron oxygenase systems that have the alpha3beta3 configuration and reveals the presence of the specific loops that interact with a neighboring subunit. The shape of the substrate-binding pocket of CARDO-O is markedly different from those of other oxygenase components involved in naphthalene and biphenyl degradation pathways. Docking simulations suggest that carbazole binds to the substrate-binding pocket in a manner suitable for catalysis of angular dioxygenation
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crystal structures of the nonreduced, reduced, and substrate-bound binary complexes of terminal oxygenase CARDO-O from Janthinobacterium sp. J3 with its electron donor, ferredoxin CARDO-F from Pseudomonas resinovorans CA10 at 1.9, 1.8, and 2.0 A resolutions, respectively. The structures provide a structure-based interpretation of intercomponent electron transfer between two Rieske [2Fe-2S] clusters of ferredoxin and oxygenase in a Rieske nonheme iron oxygenase system. Three molecules of CARDO-F bind to the subunit boundary of one CARDO-O trimeric molecule, and specific binding created by electrostatic and hydrophobic interactions with conformational changes suitably aligns the two Rieske clusters for electron transfer. Additionally, conformational changes upon binding carbazole results in the closure of a lid over the substrate-binding pocket, thereby seemingly trapping carbazole at the substrate-binding site
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crystallization of ferredoxin reductase subunit CARDO-R using the hanging-drop vapour-diffusion method with the precipitant PEG 8000 results in two crystal types. The type I crystal diffract to a maximum resolution of 2.80 A and belong to space group P42212, with unit cell parameters a, b of 158.7, c of 81.4 A. The type II crystal diffracts to 2.60 A resolution and belongs to the same space group, with unit-cell parameters a, b of 161.8, c of 79.5 A
docking simulation of dibenzo-p-dioxin to wild-type CARDO oxygenase
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comparison of crystal structures of the oxygenase and ferredoxin components to the CARDOs from Pseudomonas resinovorans CA10, Janthinobacterium sp. J3, Novosphingobium sp. KA1, and Nocardioides aromaticivorans IC177 which are grouped into classes III, III, IIA, and IIB, respectively. The comparison suggests residues in common between class IIB and class III CARDOs that are important for interactions between ferredoxin and oxygenase. In the class IIB CARDOs, these include His75 and Glu71 in ferredoxin and Lys20 and Glu357 in the oxygenase for electrostatic interactions, and Phe74 and Pro90 in ferredoxin and Trp21, Leu359, and Val367 in the oxygenase for hydrophobic interactions
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crystallization of ferredoxin component, to 2.0 A resolution, space group P41212; terminal oxygenase component, to 2.3 A resolution, space group C2
comparison of crystal structures of the oxygenase and ferredoxin components to the CARDOs from Pseudomonas resinovorans CA10, Janthinobacterium sp. J3, Novosphingobium sp. KA1, and Nocardioides aromaticivorans IC177 which are grouped into classes III, III, IIA, and IIB, respectively. The comparison suggests residues in common between class IIB and class III CARDOs that are important for interactions between ferredoxin and oxygenase. In the class IIB CARDOs, these include His75 and Glu71 in ferredoxin and Lys20 and Glu357 in the oxygenase for electrostatic interactions, and Phe74 and Pro90 in ferredoxin and Trp21, Leu359, and Val367 in the oxygenase for hydrophobic interactions
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crystal structure of ferredoxin component CarAc at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF
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crystal structures of the nonreduced, reduced, and substrate-bound binary complexes of terminal oxygenase CARDO-O from Janthinobacterium sp. J3 with its electron donor, ferredoxin CARDO-F from Pseudomonas resinovorans CA10 at 1.9, 1.8, and 2.0 A resolutions, respectively. The structures provide a structure-based interpretation of intercomponent electron transfer between two Rieske [2Fe-2S] clusters of ferredoxin and oxygenase in a Rieske nonheme iron oxygenase system. Three molecules of CARDO-F bind to the subunit boundary of one CARDO-O trimeric molecule, and specific binding created by electrostatic and hydrophobic interactions with conformational changes suitably aligns the two Rieske clusters for electron transfer. Additionally, conformational changes upon binding carbazole results in the closure of a lid over the substrate-binding pocket, thereby seemingly trapping carbazole at the substrate-binding site
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CARDO of Novosphingobium sp. KA1 consists of a terminal oxygenase, a putidaredoxin-type ferredoxin and a ferredoxin-NADH oxidoreductase. Crystallization of the ferredoxin reductase component to 1.58 A resolution, space group P32, with unit-cell parameters a = b = 92.2, c = 78.6 A
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CARDO of Novosphingobium sp. KA1 consists of a terminal oxygenase, Oxy, a putidaredoxin-type ferredoxin and a ferredoxin-NADH oxidoreductase. Crystallization of the oxygenase component to 2.1 A resolution, space group P21
comparison of crystal structures of the oxygenase and ferredoxin components to the CARDOs from Pseudomonas resinovorans CA10, Janthinobacterium sp. J3, Novosphingobium sp. KA1, and Nocardioides aromaticivorans IC177 which are grouped into classes III, III, IIA, and IIB, respectively. The comparison suggests residues in common between class IIB and class III CARDOs that are important for interactions between ferredoxin and oxygenase. In the class IIB CARDOs, these include His75 and Glu71 in ferredoxin and Lys20 and Glu357 in the oxygenase for electrostatic interactions, and Phe74 and Pro90 in ferredoxin and Trp21, Leu359, and Val367 in the oxygenase for hydrophobic interactions
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0°C 10% glycerol, 8 days, more than 90% residual activity, isolated subunit CarAa
0°C 50 mM Tris-HCl pH 7.5, 24 h, 90% residual activity, reconstitued CARDO system
0°C, 10 h, full activity, and 100 h, 93% residual activiy, ferredoxin reductase subunit CarAd
0°C, 10% glycerol, 8 days, more than 90% residual activity for isolated subunit CarAa
0°C, 50 mM Tris-HCl pH 7.5, 24 h, 90% residual activity for reconstituted CARDO system
4°C, 10% glycerol, 24 h, full activity for isolated subunit CarAa
4°C, 10% glycerol, 24 h, full activity, isolated subunit CarAa
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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recombinant protein; recombinant protein
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression of C-terminal His-tagged form of terminal oxygenase CarAaJ3 in Escherichia coli
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expression of ferredoxin subunit CarAc in Escherichia coli, His-tagged; expression of ferredoxin subunit CarAc in Escherichia coli, His-tagged; expression of terminal oxygenase CarAa in Escherichia coli in native form
expression of genes CARAaAcAd in Escherichia coli
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expression of genes CarAacd in Rhodococcus erythropolis
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expression of terminal oxygenase subunit in Rhizobium tropici
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the carAaBaBbCAc gene cluster encoding carbazole 1,9a-dioxygenase, ferredoxin reductase fdr, and antRAcAdAbAa gene cluster involved in the conversion of anthranilate to catechol are induced when strain XLDN2-5 is exposed to carbazole; the carAaBaBbCAc gene cluster encoding carbazole 1,9a-dioxygenase, ferredoxin reductase fdr, and antRAcAdAbAa gene cluster involved in the conversion of anthranilate to catechol are induced when strain XLDN2-5 is exposed to carbazole
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F275A
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mutation in oxygenase component. Decrease in activity with carbazole, dibenzo-p-dioxin, anthracene, very low activity with fluorene, fluoranthene
F275I
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mutation in oxygenase component. Decrease in activity with carbazole, very low activtiy with dibenzo-p-dioxin, anthracene, fluorene, fluoranthene
F275L
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mutation in oxygenase component. Decrease in activity with carbazole, very low activtiy with dibenzo-p-dioxin, anthracene, fluorene, fluoranthene
F275V
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mutation in oxygenase component. Decrease in activity with carbazole, very low activtiy with dibenzo-p-dioxin, anthracene, fluorene, fluoranthene
F275W
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mutation in oxygenase component. Decrease in activity with carbazole, wild-type like activity with dibenzo-p-dioxin, anthracene, fluorene, fluoranthene
F329A
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mutation in oxygenase component. Decrease in activity with carbazole, dibenzo-p-dioxin, anthracene, very low activity with fluorene, fluoranthene
F329I
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mutation in oxygenase component. Strong decrease in activity with all substrates tested
F329L
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mutation in oxygenase component. Strong decrease in activity with all substrates tested
F329V
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mutation in oxygenase component. Strong decrease in activity with all substrates tested
F329W
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mutation in oxygenase component. Strong decrease in activity with all substrates tested
I262A
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mutation in oxygenase component. Decrease in activity with carbazole, dibenzo-p-dioxin, anthracene, very low activity with fluorene, fluoranthene
I262L
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mutation in oxygenase component. Increase in activity with anthracene
I262V
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mutation in oxygenase component. Decrease in activity with carbazole, dibenzo-p-dioxin, anthracene, very low activity with fluorene, fluoranthene
I262W
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mutation in oxygenase component. Decrease in activity with carbazole, dibenzo-p-dioxin, anthracene, very low activity with fluorene, fluoranthene
Q282N
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mutation in oxygenase component. Decrease in activity with carbazole, very low activtiy with dibenzo-p-dioxin, anthracene, fluorene, fluoranthene
Q282S
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mutation in oxygenase component. Decrease in activity with carbazole, very low activtiy with dibenzo-p-dioxin, anthracene, fluorene, fluoranthene
Q282Y
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mutation in oxygenase component. Decrease in activity with carbazole, very low activtiy with dibenzo-p-dioxin, anthracene, fluorene, fluoranthene
degradation
additional information
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expression of the catalytic subunit CarAa is enhanced by induction at a lower cell concentration and temperature and over a longer time, while the expression of subunits CarAc and CarAd is inverse. Best co-expression condition tested is cell concentration at induction of 0.5 (absorbance at 600 nm), 37°C amd 4 h
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
degradation