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Literature summary for 1.14.12.22 extracted from

  • 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 (2002), Appl. Environ. Microbiol., 68, 5882-5890.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expression of ferredoxin subunit CarAc in Escherichia coli, His-tagged Pseudomonas resinovorans
expression of terminal oxygenase CarAa in Escherichia coli in native form Pseudomonas resinovorans

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0034
-
NADH CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C Pseudomonas resinovorans
0.182
-
NADPH CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C Pseudomonas resinovorans

Metals/Ions

Metals/Ions Comment Organism Structure
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 Pseudomonas resinovorans
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 Pseudomonas resinovorans
Iron terminal oxygenase subunit contains one Rieske type [2Fe2S] cluster and one mononuclear iron center in each monomer Pseudomonas resinovorans

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
13000
-
gel filtration Pseudomonas resinovorans
37000
-
gel filtration Pseudomonas resinovorans
43782
-
3 * 44000, SDS-PAGE, 3 * 43782, calculated, subunit CarAa Pseudomonas resinovorans
44000
-
3 * 44000, SDS-PAGE, 3 * 43782, calculated, subunit CarAa Pseudomonas resinovorans
132000
-
gel filtration, subunit CarAa Pseudomonas resinovorans

Organism

Organism UniProt Comment Textmining
Pseudomonas resinovorans
-
-
-
Pseudomonas resinovorans Q8GI14 ferredoxin reductase subunit CarAd
-
Pseudomonas resinovorans Q8GI16 ferredoxin subunit CarAc
-
Pseudomonas resinovorans CA10
-
-
-
Pseudomonas resinovorans CA10 Q8GI14 ferredoxin reductase subunit CarAd
-
Pseudomonas resinovorans CA10 Q8GI16 ferredoxin subunit CarAc
-

Purification (Commentary)

Purification (Comment) Organism
recombinant protein Pseudomonas resinovorans

Storage Stability

Storage Stability Organism
0°C 10% glycerol, 8 days, more than 90% residual activity, isolated subunit CarAa Pseudomonas resinovorans
0°C 50 mM Tris-HCl pH 7.5, 24 h, 90% residual activity, reconstitued CARDO system Pseudomonas resinovorans
0°C, 10 h, full activity, and 100 h, 93% residual activiy, ferredoxin reductase subunit CarAd Pseudomonas resinovorans
0°C, 10% glycerol, 8 days, more than 90% residual activity for isolated subunit CarAa Pseudomonas resinovorans
0°C, 50 mM Tris-HCl pH 7.5, 24 h, 90% residual activity for reconstituted CARDO system Pseudomonas resinovorans
4°C, 10% glycerol, 24 h, full activity for isolated subunit CarAa Pseudomonas resinovorans
4°C, 10% glycerol, 24 h, full activity, isolated subunit CarAa Pseudomonas resinovorans

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
carbazole + NADH + H+ + O2
-
Pseudomonas resinovorans 2'-aminobiphenyl-2,3-diol + NAD+
-
?
carbazole + NADH + H+ + O2
-
Pseudomonas resinovorans CA10 2'-aminobiphenyl-2,3-diol + NAD+
-
?
carbazole + NADPH + H+ + O2
-
Pseudomonas resinovorans 2'-aminobiphenyl-2,3-diol + NADP+
-
?
carbazole + NADPH + H+ + O2
-
Pseudomonas resinovorans CA10 2'-aminobiphenyl-2,3-diol + NADP+
-
?
additional information 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 Pseudomonas resinovorans ?
-
?
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 Pseudomonas resinovorans ?
-
?
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 Pseudomonas resinovorans ?
-
?
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 Pseudomonas resinovorans CA10 ?
-
?
additional information 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 Pseudomonas resinovorans CA10 ?
-
?
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 Pseudomonas resinovorans CA10 ?
-
?

Subunits

Subunits Comment Organism
monomer 1 * 13000, SDS-PAGE, subunit CarAc Pseudomonas resinovorans
monomer 1 * 37000, SDS-PAGE, subunit CarAd Pseudomonas resinovorans
trimer 3 * 44000, SDS-PAGE, 3 * 43782, calculated, subunit CarAa Pseudomonas resinovorans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
-
Pseudomonas resinovorans
35 45 maximum cytochrome c reductase activity of subunit CarAd Pseudomonas resinovorans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
-
Pseudomonas resinovorans
7.5 8.5 maximum cytochrome c reductase activity of subunit CarAd Pseudomonas resinovorans

Cofactor

Cofactor Comment Organism Structure
FAD 1 mol of His-tagged CarAd contains 1 mol of FAD Pseudomonas resinovorans
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 Pseudomonas resinovorans
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 Pseudomonas resinovorans

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
130
-
NADPH CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C Pseudomonas resinovorans
2900
-
NADH CarAd activity, electron acceptor 2,6-dichlorophenolindophenol, pH 7.5, 30°C Pseudomonas resinovorans