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Literature summary extracted from

  • Walczak, R.J.; Dickens, M.L.; Priestley, N.D.; Strohl, W.R.
    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.
    View publication on PubMedView publication on EuropePMC

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.14.13.181 dithiothreitol strongly inhibitory, inhibition is reversible after desalting Streptomyces sp.
1.14.13.181 doxorubicin completely inhibits DoxA activity at both 1 and 5 mM, competitive with respect to daunorubicinol oxidation Streptomyces sp.
1.14.13.181 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.
1.14.13.181 quinidine inhibits DoxA oxidation of daunorubicinol by 11% at 1 mM. At 5 mM, it inhibits at more than 95% Streptomyces sp.
1.14.13.181 sulfaphenazole inhibits DoxA oxidation of daunorubicinol by 20% at 1 mM. At 5 mM, it inhibits at more than 95% Streptomyces sp.
1.14.13.181 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.181 0.0009
-
daunorubicin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 0.0011
-
13-deoxydaunorubicin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 0.0025
-
13-deoxycarminomycin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 0.0046
-
13-dihydrodaunorubicin pH 7.5, 30°C Streptomyces sp.

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.14.13.181 46096
-
1 * 46096, calculated from sequence Streptomyces sp.
1.14.13.181 47000
-
1 * 47000, SDS-PAGE Streptomyces sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.181 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
-
?
1.14.13.181 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
-
?
1.14.13.181 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
-
?
1.14.13.181 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
-
?
1.14.13.181 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

EC Number Organism UniProt Comment Textmining
1.14.13.181 Streptomyces sp. Q59971
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.181 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

EC Number Source Tissue Comment Organism Textmining
1.14.13.181 mycelium
-
Streptomyces sp.
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.14.13.181 0.0006
-
pH 7.5, 30°C, 13-oxidation of daunorubicinol to daunorubicin Streptomyces sp.

Storage Stability

EC Number Storage Stability Organism
1.14.13.181 4°C, 20% (v/v) glycerol, 7 days, no appreciable loss of activity Streptomyces sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.181 13-deoxycarminomycin + NADPH + H+ + O2
-
Streptomyces sp. 13-dihydrocarminomycin + NADP+ + H2O
-
?
1.14.13.181 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
-
?
1.14.13.181 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
-
?
1.14.13.181 13-deoxydaunorubicin + NADPH + H+ + O2
-
Streptomyces sp. daunorubicinol + NADP+ + H2O
-
?
1.14.13.181 13-dihydrocarminomycin + NADPH + H+ + O2
-
Streptomyces sp. carminomycin + NADP+ + H2O
-
?
1.14.13.181 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
-
?
1.14.13.181 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
-
?
1.14.13.181 daunorubicin + NADPH + H+ + O2
-
Streptomyces sp. doxorubicin + NADP+ + H2O
-
?
1.14.13.181 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
-
?
1.14.13.181 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

EC Number Subunits Comment Organism
1.14.13.181 monomer 1 * 47000, SDS-PAGE Streptomyces sp.
1.14.13.181 monomer 1 * 46096, calculated from sequence Streptomyces sp.

Synonyms

EC Number Synonyms Comment Organism
1.14.13.181 DoxA
-
Streptomyces sp.
1.14.13.181 DoxA monooxygenase
-
Streptomyces sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.181 30
-
-
Streptomyces sp.

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.14.13.181 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]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.181 0.00012
-
daunorubicin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 0.00026
-
13-deoxycarminomycin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 0.024
-
13-deoxydaunorubicin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 0.63
-
13-dihydrodaunorubicin pH 7.5, 30°C Streptomyces sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.181 7.5
-
-
Streptomyces sp.

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.181 cytochrome P-450 cytochrome P-450-dependent monooxygenase Streptomyces sp.
1.14.13.181 NADPH
-
Streptomyces sp.

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.14.13.181 0.021
-
doxorubicin pH 7.5, 30°C Streptomyces sp.

General Information

EC Number General Information Comment Organism
1.14.13.181 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.181 0.104
-
13-deoxycarminomycin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 0.133
-
daunorubicin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 21.81
-
13-deoxydaunorubicin pH 7.5, 30°C Streptomyces sp.
1.14.13.181 136.9
-
13-dihydrodaunorubicin pH 7.5, 30°C Streptomyces sp.