BRENDA - Enzyme Database

Characterization of iron-sulfur clusters in flavin-containing opine dehydrogenase

Watanabe, S.; Tajima, K.; Matsui, K.; Watanabe, Y.; Biosci. Biotechnol. Biochem. 80, 2371-2375 (2016)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.5.99.B4
genes bll7191, bll7190, and bll7192, sequence comparisons and phylogenetic analysis, recombinant expression from vectors pETDuet-1, pACYCDuet-1, and pCOLADuet-1 with the (His)6-tag attached to the N-terminus of the bll7190 gene, whereas the S-tag is attached to the C-termini of the bll7191 and bll7192 genes, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
Bradyrhizobium japonicum
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.5.99.B4
C382S
site-directed mutagenesis, FAD and FMN are detected at very low level in the extract of the alphaC382Sbetagamma mutant. The recombinant expression level of the alphaC382Sbetagamma mutant is approximately 6fold lower than that of alphabetagamma-wild-type
Bradyrhizobium japonicum
1.5.99.B4
C61S
site-directed mutagenesis, the alphabetagammaC61S mutant is more resistant to the thermal treatment than the wild-type
Bradyrhizobium japonicum
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.5.99.B4
Fe2+
electron paramagnetic resonance analysis
Bradyrhizobium japonicum
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
Bradyrhizobium japonicum
i.e. D-octopine
L-arginine + pyruvate + FADH2
-
-
?
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
Bradyrhizobium japonicum USDA110
i.e. D-octopine
L-arginine + pyruvate + FADH2
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.5.99.B4
Bradyrhizobium japonicum
Q89E96 AND Q89E94 AND Q89E97 AND
subunits BjOdhA (bll7191 or ooxA), BjOdhB1 (bll7193), and BjOdhB2 (bll7190 or soxB)
-
1.5.99.B4
Bradyrhizobium japonicum USDA110
Q89E96 AND Q89E94 AND Q89E97 AND
subunits BjOdhA (bll7191 or ooxA), BjOdhB1 (bll7193), and BjOdhB2 (bll7190 or soxB)
-
Purification (Commentary)
EC Number
Commentary
Organism
1.5.99.B4
recombinant His- and S-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography as holoenzymes. The expression level of the alphaC382Sbetagamma mutant is approximately 6fold lower than that of alphabetagamma-wild-type
Bradyrhizobium japonicum
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [mol/min/mg]
Specific Activity Maximum [mol/min/mg]
Commentary
Organism
1.5.99.B4
0.257
-
purified recombinant enzyme mutant alphaC382Sbetagamma, pH 9.0, 30C
Bradyrhizobium japonicum
1.5.99.B4
0.504
-
purified recombinant wild-type enzyme, pH 9.0, 30C
Bradyrhizobium japonicum
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
i.e. D-octopine
742120
Bradyrhizobium japonicum
L-arginine + pyruvate + FADH2
-
-
-
?
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
i.e. D-octopine, the electrons obtained through the oxidation of octopine are received by FMN via FAD in the beta-subunit, and finally transferred to an electron acceptor via the [2Fe-2S] cluster bound to [Fe-S]site 2, FAD in the alpha-subunit, and/or [4Fe-4S] cluster bound to [Fe-S]site 1. 2,6-Dichloroindophenol is used as final electron acceptor
742120
Bradyrhizobium japonicum
L-arginine + pyruvate + FADH2
-
-
-
?
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
i.e. D-octopine
742120
Bradyrhizobium japonicum USDA110
L-arginine + pyruvate + FADH2
-
-
-
?
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
i.e. D-octopine, the electrons obtained through the oxidation of octopine are received by FMN via FAD in the beta-subunit, and finally transferred to an electron acceptor via the [2Fe-2S] cluster bound to [Fe-S]site 2, FAD in the alpha-subunit, and/or [4Fe-4S] cluster bound to [Fe-S]site 1. 2,6-Dichloroindophenol is used as final electron acceptor
742120
Bradyrhizobium japonicum USDA110
L-arginine + pyruvate + FADH2
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.5.99.B4
heterododecamer
alpha4beta4gamma4
Bradyrhizobium japonicum
Temperature Optimum [C]
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
1.5.99.B4
30
-
assay at
Bradyrhizobium japonicum
Temperature Stability [C]
EC Number
Temperature Stability Minimum [C]
Temperature Stability Maximum [C]
Commentary
Organism
1.5.99.B4
45
-
after incubation for 10 min at 45C, pH 8.0, the activities of mutant alphabetagammaC61S and alphabetagamma-wild-type maintain 49% and 30% of initial activity, respectively
Bradyrhizobium japonicum
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.5.99.B4
9
-
assay at
Bradyrhizobium japonicum
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.5.99.B4
FAD
FAD in alpha- and beta-subunits
Bradyrhizobium japonicum
1.5.99.B4
FMN
1 FMN between the alpha- and beta-subunits
Bradyrhizobium japonicum
1.5.99.B4
additional information
[4Fe-4S] and [2Fe-2S] clusters bind to two different types of [Fe-S] binding sites in the gamma- and alpha-subunits, respectively. Site [Fe-S]site 1 consists of Cys56-X4-Cys61-X2-Cys64-X11~12-Cys77 near the C-terminus, while site [Fe-S]site 2 consists of Cys380-X-Cys382-X15~20-Cys415-X4-Cys420; cysteine residues in the [Fe-S] binding site natively form disulfide bond(s)
Bradyrhizobium japonicum
1.5.99.B4
[2Fe-2S]-center
importance of the [2Fe-2S] cluster for catalytic activity, the [Fe-S]site 2 of BjOpnDH binds to the [2Fe-2S] clusters. The gamma-subunit by itself is necessary for the adequate binding of [2Fe-2S]. The [2Fe-2S] cluster is important for structural folding and enzyme catalysis
Bradyrhizobium japonicum
1.5.99.B4
[4Fe-4S]-center
the [Fe-S]site 1 of BjOpnDH binds to the [4Fe-4S] clusters
Bradyrhizobium japonicum
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.5.99.B4
genes bll7191, bll7190, and bll7192, sequence comparisons and phylogenetic analysis, recombinant expression from vectors pETDuet-1, pACYCDuet-1, and pCOLADuet-1 with the (His)6-tag attached to the N-terminus of the bll7190 gene, whereas the S-tag is attached to the C-termini of the bll7191 and bll7192 genes, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
Bradyrhizobium japonicum
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.5.99.B4
FAD
FAD in alpha- and beta-subunits
Bradyrhizobium japonicum
1.5.99.B4
FMN
1 FMN between the alpha- and beta-subunits
Bradyrhizobium japonicum
1.5.99.B4
additional information
[4Fe-4S] and [2Fe-2S] clusters bind to two different types of [Fe-S] binding sites in the gamma- and alpha-subunits, respectively. Site [Fe-S]site 1 consists of Cys56-X4-Cys61-X2-Cys64-X11~12-Cys77 near the C-terminus, while site [Fe-S]site 2 consists of Cys380-X-Cys382-X15~20-Cys415-X4-Cys420; cysteine residues in the [Fe-S] binding site natively form disulfide bond(s)
Bradyrhizobium japonicum
1.5.99.B4
[2Fe-2S]-center
importance of the [2Fe-2S] cluster for catalytic activity, the [Fe-S]site 2 of BjOpnDH binds to the [2Fe-2S] clusters. The gamma-subunit by itself is necessary for the adequate binding of [2Fe-2S]. The [2Fe-2S] cluster is important for structural folding and enzyme catalysis
Bradyrhizobium japonicum
1.5.99.B4
[4Fe-4S]-center
the [Fe-S]site 1 of BjOpnDH binds to the [4Fe-4S] clusters
Bradyrhizobium japonicum
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.5.99.B4
C382S
site-directed mutagenesis, FAD and FMN are detected at very low level in the extract of the alphaC382Sbetagamma mutant. The recombinant expression level of the alphaC382Sbetagamma mutant is approximately 6fold lower than that of alphabetagamma-wild-type
Bradyrhizobium japonicum
1.5.99.B4
C61S
site-directed mutagenesis, the alphabetagammaC61S mutant is more resistant to the thermal treatment than the wild-type
Bradyrhizobium japonicum
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.5.99.B4
Fe2+
electron paramagnetic resonance analysis
Bradyrhizobium japonicum
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
Bradyrhizobium japonicum
i.e. D-octopine
L-arginine + pyruvate + FADH2
-
-
?
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
Bradyrhizobium japonicum USDA110
i.e. D-octopine
L-arginine + pyruvate + FADH2
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.5.99.B4
recombinant His- and S-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography as holoenzymes. The expression level of the alphaC382Sbetagamma mutant is approximately 6fold lower than that of alphabetagamma-wild-type
Bradyrhizobium japonicum
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [mol/min/mg]
Specific Activity Maximum [mol/min/mg]
Commentary
Organism
1.5.99.B4
0.257
-
purified recombinant enzyme mutant alphaC382Sbetagamma, pH 9.0, 30C
Bradyrhizobium japonicum
1.5.99.B4
0.504
-
purified recombinant wild-type enzyme, pH 9.0, 30C
Bradyrhizobium japonicum
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
i.e. D-octopine
742120
Bradyrhizobium japonicum
L-arginine + pyruvate + FADH2
-
-
-
?
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
i.e. D-octopine, the electrons obtained through the oxidation of octopine are received by FMN via FAD in the beta-subunit, and finally transferred to an electron acceptor via the [2Fe-2S] cluster bound to [Fe-S]site 2, FAD in the alpha-subunit, and/or [4Fe-4S] cluster bound to [Fe-S]site 1. 2,6-Dichloroindophenol is used as final electron acceptor
742120
Bradyrhizobium japonicum
L-arginine + pyruvate + FADH2
-
-
-
?
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
i.e. D-octopine
742120
Bradyrhizobium japonicum USDA110
L-arginine + pyruvate + FADH2
-
-
-
?
1.5.99.B4
N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O
i.e. D-octopine, the electrons obtained through the oxidation of octopine are received by FMN via FAD in the beta-subunit, and finally transferred to an electron acceptor via the [2Fe-2S] cluster bound to [Fe-S]site 2, FAD in the alpha-subunit, and/or [4Fe-4S] cluster bound to [Fe-S]site 1. 2,6-Dichloroindophenol is used as final electron acceptor
742120
Bradyrhizobium japonicum USDA110
L-arginine + pyruvate + FADH2
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.5.99.B4
heterododecamer
alpha4beta4gamma4
Bradyrhizobium japonicum
Temperature Optimum [C] (protein specific)
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
1.5.99.B4
30
-
assay at
Bradyrhizobium japonicum
Temperature Stability [C] (protein specific)
EC Number
Temperature Stability Minimum [C]
Temperature Stability Maximum [C]
Commentary
Organism
1.5.99.B4
45
-
after incubation for 10 min at 45C, pH 8.0, the activities of mutant alphabetagammaC61S and alphabetagamma-wild-type maintain 49% and 30% of initial activity, respectively
Bradyrhizobium japonicum
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.5.99.B4
9
-
assay at
Bradyrhizobium japonicum
General Information
EC Number
General Information
Commentary
Organism
1.5.99.B4
evolution
two types of ProDHs, alphabetagammadelta, and alpha4beta4 complexes, have been identified from hyperthermophilic archaea, and their alpha-subunits commonly correspond to the gamma-subunit of OpnDH. OpnDH belongs to a group of so-called dye-linked dehydrogenases that catalyze the oxidation of various organic acids, amino acids, and alcohols in the presence of an artificial electron acceptor, such as 2,6-dichloroindophenol, in which FAD and/or FMN is commonly contained as a prosthetic group(s)
Bradyrhizobium japonicum
1.5.99.B4
malfunction
loss of the [4Fe-4S] cluster and/or gamma-subunit by itself has no effect on the binding of FAD and FMN. A marked decrease in the activity of the alphabeta mutant appears to be due to the (partial) degradation of the [2Fe-2S] cluster
Bradyrhizobium japonicum
1.5.99.B4
additional information
the gamma-subunit by itself is necessary for the adequate binding of FMN and [2Fe-2S], particularly the latter. The removal of the [4Fe-4S] cluster may result in the easier formation of a disulfide bond between the remaining three cysteine residues in [Fe-S]site 1 of the alphabetagammaC61S mutant than in alphabetagamma-wild-type for structural stabilization
Bradyrhizobium japonicum
General Information (protein specific)
EC Number
General Information
Commentary
Organism
1.5.99.B4
evolution
two types of ProDHs, alphabetagammadelta, and alpha4beta4 complexes, have been identified from hyperthermophilic archaea, and their alpha-subunits commonly correspond to the gamma-subunit of OpnDH. OpnDH belongs to a group of so-called dye-linked dehydrogenases that catalyze the oxidation of various organic acids, amino acids, and alcohols in the presence of an artificial electron acceptor, such as 2,6-dichloroindophenol, in which FAD and/or FMN is commonly contained as a prosthetic group(s)
Bradyrhizobium japonicum
1.5.99.B4
malfunction
loss of the [4Fe-4S] cluster and/or gamma-subunit by itself has no effect on the binding of FAD and FMN. A marked decrease in the activity of the alphabeta mutant appears to be due to the (partial) degradation of the [2Fe-2S] cluster
Bradyrhizobium japonicum
1.5.99.B4
additional information
the gamma-subunit by itself is necessary for the adequate binding of FMN and [2Fe-2S], particularly the latter. The removal of the [4Fe-4S] cluster may result in the easier formation of a disulfide bond between the remaining three cysteine residues in [Fe-S]site 1 of the alphabetagammaC61S mutant than in alphabetagamma-wild-type for structural stabilization
Bradyrhizobium japonicum
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.5.99.B4
1.97
-
N2-(D-1-carboxyethyl)-L-arginine
mutant alphaC382Sbetagamma, pH 9.0, 30C
Bradyrhizobium japonicum
1.5.99.B4
3.73
-
N2-(D-1-carboxyethyl)-L-arginine
recombinant wild-type enzyme, pH 9.0, 30C
Bradyrhizobium japonicum
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.5.99.B4
1.97
-
N2-(D-1-carboxyethyl)-L-arginine
mutant alphaC382Sbetagamma, pH 9.0, 30C
Bradyrhizobium japonicum
1.5.99.B4
3.73
-
N2-(D-1-carboxyethyl)-L-arginine
recombinant wild-type enzyme, pH 9.0, 30C
Bradyrhizobium japonicum