BRENDA - Enzyme Database

Characterization of 3-ketosteroid 9alpha-hydroxylase, a Rieske oxygenase in the cholesterol degradation pathway of Mycobacterium tuberculosis

Capyk, J.; DAngelo, I.; Strynadka, N.; Eltis, L.; J. Biol. Chem. 284, 9937-9946 (2009)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.14.15.30
expressed in Escherichia coli BL21(DE3) cells
Mycobacterium tuberculosis
Crystallization (Commentary)
EC Number
Crystallization
Organism
1.14.15.30
component KshA, sitting drop vapor diffusion method, using 1.1 M sodium malonate, 0.1 M HEPES, pH 7.0, and 0.5% (v/v) Jeffamine M600
Mycobacterium tuberculosis
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.15.30
0.024
-
4-androstene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
0.11
-
1,4-androstadiene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
1.2
-
O2
in the presence of 1,4-androstadiene-3,17-dione, Km above 1.2 mM, in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.14.15.30
Mycobacterium tuberculosis
P71875
-
-
1.14.15.30
Mycobacterium tuberculosis H37Rv
P71875
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.14.15.30
Ni2+-NTA agarose column chromatography and Source15Q column chromatography
Mycobacterium tuberculosis
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
best substrate, the substrate is twice as efficient as : 4-androstene-3,17-dione
717784
Mycobacterium tuberculosis
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
ir
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
best substrate, the substrate is twice as efficient as : 4-androstene-3,17-dione
717784
Mycobacterium tuberculosis H37Rv
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
ir
1.14.15.30
4-androstene-3,17-dione + O2 + NADH + H+
-
717784
Mycobacterium tuberculosis
9alpha-hydroxy-4-androstene-3,17-dione + H2O + NAD+
-
-
-
-
1.14.15.30
4-androstene-3,17-dione + O2 + NADH + H+
-
717784
Mycobacterium tuberculosis H37Rv
9alpha-hydroxy-4-androstene-3,17-dione + H2O + NAD+
-
-
-
-
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.14.15.30
0.07
-
4-androstene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
0.8
-
1,4-androstadiene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
7
-
O2
in the presence of 1,4-androstadiene-3,17-dione, kcat above 7 1/sec, in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.14.15.30
[2Fe-2S]-center
8.2 mol of iron and 2.4 mol of sulfur per mol of KshA protomer
Mycobacterium tuberculosis
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.14.15.30
expressed in Escherichia coli BL21(DE3) cells
Mycobacterium tuberculosis
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.14.15.30
[2Fe-2S]-center
8.2 mol of iron and 2.4 mol of sulfur per mol of KshA protomer
Mycobacterium tuberculosis
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
1.14.15.30
component KshA, sitting drop vapor diffusion method, using 1.1 M sodium malonate, 0.1 M HEPES, pH 7.0, and 0.5% (v/v) Jeffamine M600
Mycobacterium tuberculosis
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.15.30
0.024
-
4-androstene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
0.11
-
1,4-androstadiene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
1.2
-
O2
in the presence of 1,4-androstadiene-3,17-dione, Km above 1.2 mM, in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.14.15.30
Ni2+-NTA agarose column chromatography and Source15Q column chromatography
Mycobacterium tuberculosis
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
best substrate, the substrate is twice as efficient as : 4-androstene-3,17-dione
717784
Mycobacterium tuberculosis
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
ir
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
best substrate, the substrate is twice as efficient as : 4-androstene-3,17-dione
717784
Mycobacterium tuberculosis H37Rv
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
ir
1.14.15.30
4-androstene-3,17-dione + O2 + NADH + H+
-
717784
Mycobacterium tuberculosis
9alpha-hydroxy-4-androstene-3,17-dione + H2O + NAD+
-
-
-
-
1.14.15.30
4-androstene-3,17-dione + O2 + NADH + H+
-
717784
Mycobacterium tuberculosis H37Rv
9alpha-hydroxy-4-androstene-3,17-dione + H2O + NAD+
-
-
-
-
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.14.15.30
0.07
-
4-androstene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
0.8
-
1,4-androstadiene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
7
-
O2
in the presence of 1,4-androstadiene-3,17-dione, kcat above 7 1/sec, in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.14.15.30
2.45
-
O2
in the presence of 1,4-androstadiene-3,17-dione, in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
3
-
4-androstene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
7.6
-
1,4-androstadiene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
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
1.14.15.30
2.45
-
O2
in the presence of 1,4-androstadiene-3,17-dione, in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
3
-
4-androstene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis
1.14.15.30
7.6
-
1,4-androstadiene-3,17-dione
in 0.1 M potassium phosphate, pH 7.0, at 25C
Mycobacterium tuberculosis