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Literature summary for 1.14.13.181 extracted from
- Purification, properties, and characterization of recombinant Streptomyces sp. strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in doxorubicin biosynthesis (1999), J. Bacteriol., 181, 298-304.
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| dithiothreitol | strongly inhibitory, inhibition is reversible after desalting | Streptomyces sp. |  |
| doxorubicin | completely inhibits DoxA activity at both 1 and 5 mM, competitive with respect to daunorubicinol oxidation | Streptomyces sp. | |
| additional information | high ionic strength buffers containing 100 mM sodium phosphate, also strongly inhibits DoxA activity. This effect is partially reversible after exchange into low ionic strength buffers, such as 20 mM N-(2-hydroxyethyl)piperazine-N9-(2-ethanesulfonic acid) (HEPES) or 10 mM sodium phosphate (pH 7.5). No inhibition: 4-methylpyrazole (1 or 5 mM), rhodomycin D (1-5 mM) | Streptomyces sp. | |
| quinidine | inhibits DoxA oxidation of daunorubicinol by 11% at 1 mM. At 5 mM, it inhibits at more than 95% | Streptomyces sp. | |
| sulfaphenazole | inhibits DoxA oxidation of daunorubicinol by 20% at 1 mM. At 5 mM, it inhibits at more than 95% | Streptomyces sp. | |
| troleandomycin | inhibits DoxA oxidation of daunorubicinol by 35% at 1 mM. At 5 mM, it inhibits at more than 95% | Streptomyces sp. | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0009 | - |
daunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
| 0.0011 | - |
13-deoxydaunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
| 0.0025 | - |
13-deoxycarminomycin | pH 7.5, 30°C | Streptomyces sp. | |
| 0.0046 | - |
13-dihydrodaunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 46096 | - |
1 * 46096, calculated from sequence | Streptomyces sp. |
| 47000 | - |
1 * 47000, SDS-PAGE | Streptomyces sp. |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 13-deoxycarminomycin + NADPH + H+ + O2 | Streptomyces sp. | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | 13-dihydrocarminomycin + NADP+ + H2O | - |
? | |
| 13-deoxydaunorubicin + NADPH + H+ + O2 | Streptomyces sp. | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | 13-dihydrodaunorubicin + NADP+ + H2O | - |
? | |
| 13-dihydrocarminomycin + NADPH + H+ + O2 | Streptomyces sp. | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | carminomycin + NADP+ + H2O | - |
? | |
| 13-dihydrodaunorubicin + NADPH + H+ + O2 | Streptomyces sp. | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | daunorubicin + NADP+ + H2O | - |
? | |
| daunorubicin + NADPH + H+ + O2 | Streptomyces sp. | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | doxorubicin + NADP+ + H2O | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Streptomyces sp. | Q59971 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| the recombinant DoxA is purified to homogeneity from Streptomyces lividans transformed with a plasmid containing the Streptomyces sp. strain C5 doxA gene under the control of the strong SnpR-activated snpA promoter | Streptomyces sp. |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| mycelium | - |
Streptomyces sp. | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.0006 | - |
pH 7.5, 30°C, 13-oxidation of daunorubicinol to daunorubicin | Streptomyces sp. |
Storage Stability
| Storage Stability | Organism |
|---|---|
| 4°C, 20% (v/v) glycerol, 7 days, no appreciable loss of activity | Streptomyces sp. |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 13-deoxycarminomycin + NADPH + H+ + O2 | - |
Streptomyces sp. | 13-dihydrocarminomycin + NADP+ + H2O | - |
? | |
| 13-deoxycarminomycin + NADPH + H+ + O2 | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | Streptomyces sp. | 13-dihydrocarminomycin + NADP+ + H2O | - |
? | |
| 13-deoxydaunorubicin + NADPH + H+ + O2 | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | Streptomyces sp. | 13-dihydrodaunorubicin + NADP+ + H2O | - |
? | |
| 13-deoxydaunorubicin + NADPH + H+ + O2 | - |
Streptomyces sp. | daunorubicinol + NADP+ + H2O | - |
? | |
| 13-dihydrocarminomycin + NADPH + H+ + O2 | - |
Streptomyces sp. | carminomycin + NADP+ + H2O | - |
? | |
| 13-dihydrocarminomycin + NADPH + H+ + O2 | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | Streptomyces sp. | carminomycin + NADP+ + H2O | - |
? | |
| 13-dihydrodaunorubicin + NADPH + H+ + O2 | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | Streptomyces sp. | daunorubicin + NADP+ + H2O | - |
? | |
| daunorubicin + NADPH + H+ + O2 | - |
Streptomyces sp. | doxorubicin + NADP+ + H2O | - |
? | |
| daunorubicin + NADPH + H+ + O2 | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and daunorubicinol and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | Streptomyces sp. | doxorubicin + NADP+ + H2O | - |
? | |
| additional information | the enzyme shows broad substrate specificity for anthracycline glycone substrates. DoxA has a strong preference for 4-methoxyanthracycline intermediates over their 4-hydroxy analogues as well as a preference for hydroxylation of the C-13 position over the C-14 position | Streptomyces sp. | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| monomer | 1 * 47000, SDS-PAGE | Streptomyces sp. |
| monomer | 1 * 46096, calculated from sequence | Streptomyces sp. |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| DoxA | - |
Streptomyces sp. |
| DoxA monooxygenase | - |
Streptomyces sp. |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
- |
Streptomyces sp. |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 22 | - |
stable for 1 h in presence of 20% (vol/vol) glycerol. All activity is lost after 30 min at room temperature in the absence of glycerol | Streptomyces sp. |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.00012 | - |
daunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
| 0.00026 | - |
13-deoxycarminomycin | pH 7.5, 30°C | Streptomyces sp. | |
| 0.024 | - |
13-deoxydaunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
| 0.63 | - |
13-dihydrodaunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
- |
Streptomyces sp. |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| cytochrome P-450 | cytochrome P-450-dependent monooxygenase | Streptomyces sp. | |
| NADPH | - |
Streptomyces sp. | |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.021 | - |
doxorubicin | pH 7.5, 30°C | Streptomyces sp. | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| physiological function | DoxA catalyzes three steps in the daunorubicin/doxorubicin biosynthesis pathway: hydroxylation at C13 of 13-deoxycarminomycin and 13-deoxydaunorubicin, oxidation at C-13 of 13-dihydrocarminomycin and 13-dihydrodaunorubicin and the hydroxylation at C-14 of daunorubicin. It appears that the primary function of DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to daunorubicin via daunorubicinol | Streptomyces sp. |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.104 | - |
13-deoxycarminomycin | pH 7.5, 30°C | Streptomyces sp. | |
| 0.133 | - |
daunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
| 21.81 | - |
13-deoxydaunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
| 136.9 | - |
13-dihydrodaunorubicin | pH 7.5, 30°C | Streptomyces sp. | |
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