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Information on EC 1.14.12.22 - carbazole 1,9a-dioxygenase and Organism(s) Pseudomonas resinovorans and UniProt Accession Q8GI14
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1.14.12.22 carbazole 1,9a-dioxygenaseIUBMB Comments
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.
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Word Map
- 1.14.12.22
- ferredoxin
-
rieske
- resinovorans
- carac
-
carbazole-degradation
- dibenzo-p-dioxins
- dibenzofurans
- fluorene
- aromaticivorans
-
nocardioides
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dianhydride
- dibenzothiophene
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polyimide
- janthinobacterium
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stipes
- degradation
The taxonomic range for the selected organisms is: Pseudomonas resinovorans
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
cardo, carbazole 1,9a-dioxygenase, caraa, carbazole dioxygenase, cardo-f, cardo-r, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CARDO
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-
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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
194812-10-7
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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
carbazole + NADH + H+ + O2
2'-aminobiphenyl-2,3-diol + NAD+
-
-
-
?
carbazole + NADPH + H+ + O2
2'-aminobiphenyl-2,3-diol + NADP+
-
-
-
?
9-fluorenone + NAD(P)H + H+ + O2
1,1a-dihydroxy-1-hydrofluoren-9-one + NAD(P)+
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-
angular dioxygenation, yield 8-12%
-
?
biphenyl + NAD(P)H + H+ + O2
cis-2,3-dihydroxy-2,3-dihydrobiphenyl + 2-hydroxybiphenyl + 3-hydroxybiphenyl + NAD(P)+
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-
lateral dioxygenation to cis-2,3-dihydroxy-2,3-dihydrobiphenyl, yield 85-90%, and to dihydrodiol, yield 9-11%
-
?
carbazole + NAD(P)H + H+ + O2
2'-aminobiphenyl-2,3-diol + NAD(P)+
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best substrate
angular dioxygenation, yield 70-80%
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?
dibenzofuran + NAD(P)H + H+ + O2
2,2',3-trihydroxybiphenyl + NAD(P)+
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-
angular dioxygenation, yield 60-65%
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?
dibenzothiophene + NADH + H+ + O2
dibenzothiophene sulfoxide + NAD+ + H2O
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-
monooxygenation, yield 99-100%
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?
fluorene + NAD(P)H + H+ + O2
9-fluorenol + ? + NAD(P)+
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monooxygenation to 9-fluorenol, yield 3-5%, and lateral dioxygenation to dihydrodiol, yield 5-8%
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?
naphthalene + NAD(P)H + H+ + O2
cis-1,2-dihydroxy-1,2-dihydronaphthalene + 1-naphthol + NAD(P)+
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lateral dioxygenation, yield 65-70%
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?
additional information
?
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in the presence of NADH, His-tagged ferrdoxin subunit CarAc is reduced by His-tagged ferredoxin reductase CarAd. Terminal oxygenase subunit CarAa is reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins CarAa, CarAc and CarAd can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
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-
?
additional information
?
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in the presence of NADH, His-tagged ferrdoxin subunit CarAc is reduced by His-tagged ferredoxin reductase CarAd. Terminal oxygenase subunit CarAa is reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins CarAa, CarAc and CarAd can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
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-
?
additional information
?
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in the presence of NADH, His-tagged ferrdoxin subunit CarAc is reduced by His-tagged ferredoxin reductase CarAd. Terminal oxygenase subunit CarAa is reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins CarAa, CarAc and CarAd can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
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-
?
additional information
?
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in the presence of NADH, His-tagged ferrdoxin subunit CarAc is reduced by His-tagged ferredoxin reductase CarAd. The three purified proteins CarAa, CarAc and CarAd can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
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-
?
additional information
?
-
in the presence of NADH, His-tagged ferrdoxin subunit CarAc is reduced by His-tagged ferredoxin reductase CarAd. The three purified proteins CarAa, CarAc and CarAd can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
-
-
?
additional information
?
-
in the presence of NADH, His-tagged ferrdoxin subunit CarAc is reduced by His-tagged ferredoxin reductase CarAd. The three purified proteins CarAa, CarAc and CarAd can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
-
-
?
additional information
?
-
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terminal oxygenase subunit CarAa is reduced by His-tagged ferredoxin CarAc, His-tagged ferredoxin reductase CarAd, and NADH. The three purified proteins can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
-
-
?
additional information
?
-
terminal oxygenase subunit CarAa is reduced by His-tagged ferredoxin CarAc, His-tagged ferredoxin reductase CarAd, and NADH. The three purified proteins can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
-
-
?
additional information
?
-
terminal oxygenase subunit CarAa is reduced by His-tagged ferredoxin CarAc, His-tagged ferredoxin reductase CarAd, and NADH. The three purified proteins can reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc is indispensable for electron transport, while His-tagged CarAd can be replaced by some unrelated reductases
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
Ferredoxin
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ferredoxin component CarAc acts as a mediator in the electron transfer from ferredoxin reductase CarAd to terminal oxygenase CarAa. It contains a Rieske [2Fe-2S] cluster, which is located at the tip of the cluster-binding domain, where it is exposed to solvent
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Ferredoxin
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ferredoxin component contains two Rieske [2Fe-2S] clusters
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Iron
subunit CarAd, one Rieske type [2Fe-2S] cluster per monomer protein. The iron and sulfur contents of His-tagged CarAc are 1.8 to 2.0 and 1.8 to 1.9 mol/mol of protein, respectively
Iron
Iron
subunit CarAc, one Rieske type [2Fe-2S] cluster per monomer protein. The iron and sulfur contents of His-tagged CarAc are 1.7 to 1.9 and 1.3 to 1.6 mol/mol of protein, respectively
Iron
terminal oxygenase subunit contains one Rieske type [2Fe2S] cluster and one mononuclear iron center in each monomer
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
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
LITERATURE
IMAGE
2900
NADH
CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C
130
NADPH
CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5 - 8.5
maximum cytochrome c reductase activity of subunit CarAd
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
35 - 45
maximum cytochrome c reductase activity of subunit CarAd
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
43782
3 * 44000, SDS-PAGE, 3 * 43782, calculated, subunit CarAa
44000
3 * 44000, SDS-PAGE, 3 * 43782, calculated, subunit CarAa
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
trimer
3 * 44000, SDS-PAGE, 3 * 43782, calculated, subunit CarAa
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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, isolated subunit CarAa
0°C 50 mM Tris-HCl pH 7.5, 24 h, 90% residual activity, reconstitued CARDO system
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
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Nam, J.-W.; Nojiri, H.; Noguchi, H.; Uchimura, H.; Yoshida, T.; Habe, H.; Yamane, H.; Omori, T.
Purification and characterization of carbazole 1,9a-dioxygenase, a three-component dioxygenase system of Pseudomonas resinovorans strain CA10
Appl. Environ. Microbiol.
68
5882-5890
2002
Pseudomonas resinovorans, Pseudomonas resinovorans (Q8GI14), Pseudomonas resinovorans (Q8GI16), Pseudomonas resinovorans CA10, Pseudomonas resinovorans CA10 (Q8GI14), Pseudomonas resinovorans CA10 (Q8GI16)
Takagi, T.; Nojiri, H.; Yoshida, T.; Habe, H.; Omori, T.
Detailed comparison between the substrate specificities of two angular dioxygenases, dibenzofuran 4,4a-dioxygenase from Terrabacter sp. and carbazole 1,9a-dioxygenase from Pseudomonas resinovorans
Biotechnol. Lett.
24
2099-2106
2002
Pseudomonas resinovorans
-
Inoue, K.; Ashikawa, Y.; Umeda, T.; Abo, M.; Katsuki, J.; Usami, Y.; Noguchi, H.; Fujimoto, Z.; Terada, T.; Yamane, H.; Nojiri, H.
Specific Interactions between the ferredoxin and terminal oxygenase components of a class IIB Rieske nonheme iron oxygenase, carbazole 1,9a-dioxygenase
J. Mol. Biol.
392
436-451
2009
Janthinobacterium sp. J3, Nocardioides aromaticivorans, Nocardioides aromaticivorans IC177, Pseudomonas resinovorans, Pseudomonas resinovorans CA10, Sphingomonas sp., Sphingomonas sp. KA1
Nam, J.W.; Noguchi, H.; Fujimoto, Z.; Mizuno, H.; Ashikawa, Y.; Abo, M.; Fushinobu, S.; Kobashi, N.; Wakagi, T.; Iwata, K.; Yoshida, T.; Habe, H.; Yamane, H.; Omori, T.; Nojiri, H.
Crystal structure of the ferredoxin component of carbazole 1,9a-dioxygenase of Pseudomonas resinovorans strain CA10, a novel Rieske non-heme iron oxygenase system
Proteins
58
779-789
2005
Pseudomonas resinovorans, Pseudomonas resinovorans CA10
Ashikawa, Y.; Fujimoto, Z.; Noguchi, H.; Habe, H.; Omori, T.; Yamane, H.; Nojiri, H.
Electron transfer complex formation between oxygenase and ferredoxin components in Rieske nonheme iron oxygenase system
Structure
14
1779-1789
2006
Janthinobacterium sp. J3, Pseudomonas resinovorans, Pseudomonas resinovorans CA10
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