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Literature summary for 1.5.99.B4 extracted from
- Characterization of iron-sulfur clusters in flavin-containing opine dehydrogenase (2016), Biosci. Biotechnol. Biochem., 80, 2371-2375 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| 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 |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| 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 |
| C61S | site-directed mutagenesis, the alphabetagammaC61S mutant is more resistant to the thermal treatment than the wild-type | Bradyrhizobium japonicum |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Fe2+ | electron paramagnetic resonance analysis | Bradyrhizobium japonicum |  |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O | Bradyrhizobium japonicum | i.e. D-octopine | L-arginine + pyruvate + FADH2 | - |
? | |
| N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O | Bradyrhizobium japonicum USDA110 | i.e. D-octopine | L-arginine + pyruvate + FADH2 | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Bradyrhizobium japonicum | Q89E96 AND Q89E94 AND Q89E97 | subunits BjOdhA (bll7191 or ooxA), BjOdhB1 (bll7193), and BjOdhB2 (bll7190 or soxB) | - |
| Bradyrhizobium japonicum USDA110 | Q89E96 AND Q89E94 AND Q89E97 | subunits BjOdhA (bll7191 or ooxA), BjOdhB1 (bll7193), and BjOdhB2 (bll7190 or soxB) | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| 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]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.257 | - |
purified recombinant enzyme mutant alphaC382Sbetagamma, pH 9.0, 30°C | Bradyrhizobium japonicum |
| 0.504 | - |
purified recombinant wild-type enzyme, pH 9.0, 30°C | Bradyrhizobium japonicum |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O | i.e. D-octopine | Bradyrhizobium japonicum | L-arginine + pyruvate + FADH2 | - |
? | |
| 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 | Bradyrhizobium japonicum | L-arginine + pyruvate + FADH2 | - |
? | |
| N2-(D-1-carboxyethyl)-L-arginine + FAD + H2O | i.e. D-octopine | Bradyrhizobium japonicum USDA110 | L-arginine + pyruvate + FADH2 | - |
? | |
| 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 | Bradyrhizobium japonicum USDA110 | L-arginine + pyruvate + FADH2 | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| heterododecamer | alpha4beta4gamma4 | Bradyrhizobium japonicum |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| BjOpnDH | - |
Bradyrhizobium japonicum |
| bll7190 | - |
Bradyrhizobium japonicum |
| bll7191 | - |
Bradyrhizobium japonicum |
| bll7192 | - |
Bradyrhizobium japonicum |
| bll7193 | - |
Bradyrhizobium japonicum |
| flavin-containing opine dehydrogenase | - |
Bradyrhizobium japonicum |
| Nox/Oox-like protein | - |
Bradyrhizobium japonicum |
| OpnDH | - |
Bradyrhizobium japonicum |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
assay at | Bradyrhizobium japonicum |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 45 | - |
after incubation for 10 min at 45°C, pH 8.0, the activities of mutant alphabetagammaC61S and alphabetagamma-wild-type maintain 49% and 30% of initial activity, respectively | Bradyrhizobium japonicum |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 9 | - |
assay at | Bradyrhizobium japonicum |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | FAD in alpha- and beta-subunits | Bradyrhizobium japonicum | |
| FMN | 1 FMN between the alpha- and beta-subunits | Bradyrhizobium japonicum | |
| additional information | cysteine residues in the [Fe-S] binding site natively form disulfide bond(s) | Bradyrhizobium japonicum | |
| 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 | Bradyrhizobium japonicum | |
| [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 | |
| [4Fe-4S]-center | the [Fe-S]site 1 of BjOpnDH binds to the [4Fe-4S] clusters | Bradyrhizobium japonicum | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| 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 |
| 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 |
| 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]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.97 | - |
N2-(D-1-carboxyethyl)-L-arginine | mutant alphaC382Sbetagamma, pH 9.0, 30°C | Bradyrhizobium japonicum | |
| 3.73 | - |
N2-(D-1-carboxyethyl)-L-arginine | recombinant wild-type enzyme, pH 9.0, 30°C | Bradyrhizobium japonicum | |
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Release 2023.1 (January 2023)
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