BRENDA - Enzyme Database

Characterization and engineering of 3-ketosteroid-Delta1-dehydrogenase and 3-ketosteroid-9alpha-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9alpha-hydroxy-4-androstene-3,17-dione through the catabolism of sterols

Yao, K.; Xu, L.Q.; Wang, F.Q.; Wei, D.Z.; Metab. Eng. 24, 181-191 (2014)

Data extracted from this reference:

Application
EC Number
Application
Commentary
Organism
1.3.99.4
synthesis
inactivation of KstD isoforms 1-3 leads to synthesis of about 6.02g/l of 9alpha-hydroxy-4-androstene-3,17-dione from 15 g/l of phytosterols. The product is mixed with 1.55 g/l of 4-androstene-3,17-dione as a major byproduct. Overexpression of the oxygenase component of 3-ketosteroid-9alpha-hydroxylase kshA, results in a yield of 9alpha-hydroxy-4-androstene-3,17-dione of 7.33 g/l; inactivation of KstD isoforms 1-3 leads to synthesis of about 6.02g/l of 9alpha-hydroxy-4-androstene-3,17-dione from 15 g/l of phytosterols. The product is mixed with 1.55 g/l of 4-androstene-3,17-dione as a major byproduct. Overexpression of the oxygenase component of 3-ketosteroid-9alpha-hydroxylase kshA, results in a yield of 9alpha-hydroxy-4-androstene-3,17-dione of 7.33 g/l; inactivation of KstD isoforms 1-3 leads to synthesis of about 6.02g/l of 9alpha-hydroxy-4-androstene-3,17-dione from 15 g/l of phytosterols. The product is mixed with 1.55 g/l of 4-androstene-3,17-dione as a major byproduct. Overexpression of the oxygenase component of 3-ketosteroid-9alpha-hydroxylase kshA, results in a yield of 9alpha-hydroxy-4-androstene-3,17-dione of 7.33 g/l
Mycolicibacterium neoaurum
Cloned(Commentary)
EC Number
Commentary
Organism
1.3.99.4
-
Mycolicibacterium neoaurum
1.14.15.30
expressed in Escherichia coli BL21(DE3) cells
Mycolicibacterium neoaurum
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.14.15.30
additional information
recombinant coexpression of the enzyme KshA from Mycobacterium neoaurum strain NwIB-01 and the three homologues of ketosteroid-DELTA1-dehydrogenase (KstD) from Mycobacterium neoaurum ATCC257953 in Escherichia coli strain BL21(DE3)
Mycolicibacterium neoaurum
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.3.99.4
0.011
-
4-androstene-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.025
-
4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.025
-
9alpha-hydroxy-4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.035
-
5alpha-androstane-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.0379
-
5alpha-androstane-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.039
-
progesterone
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.045
-
9alpha-hydroxy-4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.06
-
progesterone
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.065
-
4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.082
-
4-androstene-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.085
-
4-androstene-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.105
-
9alpha-hydroxy-4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.125
-
progesterone
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.16
-
4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.14.15.30
Fe2+
a Rieske 2Fe-2S enzyme
Mycolicibacterium neoaurum
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
Mycolicibacterium neoaurum
-
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
Mycolicibacterium neoaurum NwIB-01
-
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
Mycolicibacterium neoaurum ATCC 25795
-
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.3.99.4
Mycolicibacterium neoaurum
C8CJG6
isoform KstD1
-
1.3.99.4
Mycolicibacterium neoaurum
W0NR13
isoform KstD2
-
1.3.99.4
Mycolicibacterium neoaurum
W0NUN5
isoform KstD3
-
1.3.99.4
Mycolicibacterium neoaurum ATCC 25795
C8CJG6
isoform KstD1
-
1.3.99.4
Mycolicibacterium neoaurum ATCC 25795
W0NR13
isoform KstD2
-
1.3.99.4
Mycolicibacterium neoaurum ATCC 25795
W0NUN5
isoform KstD3
-
1.14.15.30
Mycolicibacterium neoaurum
C9E9N6
-
-
1.14.15.30
Mycolicibacterium neoaurum ATCC 25795
C9E9N6
-
-
1.14.15.30
Mycolicibacterium neoaurum NwIB-01
C9E9N6
-
-
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.3.99.4
4-androstene-17beta-ol-3-one + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
1,4-androstadiene-17beta-ol-3-one + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
4-androstene-17beta-ol-3-one + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
1,4-androstadiene-17beta-ol-3-one + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
1,4-androstadiene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
1,4-androstadiene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
5alpha-androstane-17beta-ol-3-one + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
5alpha-androst-1-ene-17beta-ol-3-one + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
5alpha-androstane-17beta-ol-3-one + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
5alpha-androst-1-ene-17beta-ol-3-one + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
9alpha-hydroxy-4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
9alpha-hydroxyandrost-1,4-diene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
9alpha-hydroxy-4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
9alpha-hydroxyandrost-1,4-diene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
progesterone + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
pregna-1,4-diene-3,20-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
progesterone + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
pregna-1,4-diene-3,20-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
743221
Mycolicibacterium neoaurum
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
743221
Mycolicibacterium neoaurum NwIB-01
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
743221
Mycolicibacterium neoaurum ATCC 25795
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.3.99.4
?
x * 54300, calculated; x * 60100, calculated; x * 61000, calculated
Mycolicibacterium neoaurum
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.14.15.30
30
-
assay at
Mycolicibacterium neoaurum
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.14.15.30
7
-
assay at
Mycolicibacterium neoaurum
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.14.15.30
[2Fe-2S]-center
a Rieske 2Fe-2S enzyme
Mycolicibacterium neoaurum
Application (protein specific)
EC Number
Application
Commentary
Organism
1.3.99.4
synthesis
inactivation of KstD isoforms 1-3 leads to synthesis of about 6.02g/l of 9alpha-hydroxy-4-androstene-3,17-dione from 15 g/l of phytosterols. The product is mixed with 1.55 g/l of 4-androstene-3,17-dione as a major byproduct. Overexpression of the oxygenase component of 3-ketosteroid-9alpha-hydroxylase kshA, results in a yield of 9alpha-hydroxy-4-androstene-3,17-dione of 7.33 g/l
Mycolicibacterium neoaurum
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.3.99.4
-
Mycolicibacterium neoaurum
1.14.15.30
expressed in Escherichia coli BL21(DE3) cells
Mycolicibacterium neoaurum
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.14.15.30
[2Fe-2S]-center
a Rieske 2Fe-2S enzyme
Mycolicibacterium neoaurum
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.14.15.30
additional information
recombinant coexpression of the enzyme KshA from Mycobacterium neoaurum strain NwIB-01 and the three homologues of ketosteroid-DELTA1-dehydrogenase (KstD) from Mycobacterium neoaurum ATCC257953 in Escherichia coli strain BL21(DE3)
Mycolicibacterium neoaurum
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.3.99.4
0.011
-
4-androstene-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.025
-
4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.025
-
9alpha-hydroxy-4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.035
-
5alpha-androstane-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.0379
-
5alpha-androstane-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.039
-
progesterone
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.045
-
9alpha-hydroxy-4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.06
-
progesterone
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.065
-
4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.082
-
4-androstene-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.085
-
4-androstene-17beta-ol-3-one
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.105
-
9alpha-hydroxy-4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.125
-
progesterone
pH 7.0, 30°C
Mycolicibacterium neoaurum
1.3.99.4
0.16
-
4-androstene-3,17-dione
pH 7.0, 30°C
Mycolicibacterium neoaurum
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.14.15.30
Fe2+
a Rieske 2Fe-2S enzyme
Mycolicibacterium neoaurum
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
Mycolicibacterium neoaurum
-
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
Mycolicibacterium neoaurum NwIB-01
-
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
Mycolicibacterium neoaurum ATCC 25795
-
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
?
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.3.99.4
4-androstene-17beta-ol-3-one + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
1,4-androstadiene-17beta-ol-3-one + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
4-androstene-17beta-ol-3-one + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
1,4-androstadiene-17beta-ol-3-one + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
1,4-androstadiene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
1,4-androstadiene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
5alpha-androstane-17beta-ol-3-one + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
5alpha-androst-1-ene-17beta-ol-3-one + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
5alpha-androstane-17beta-ol-3-one + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
5alpha-androst-1-ene-17beta-ol-3-one + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
9alpha-hydroxy-4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
9alpha-hydroxyandrost-1,4-diene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
9alpha-hydroxy-4-androstene-3,17-dione + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
9alpha-hydroxyandrost-1,4-diene-3,17-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
progesterone + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum
pregna-1,4-diene-3,20-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.3.99.4
progesterone + 2,6-dichlorophenol-indophenol
-
743221
Mycolicibacterium neoaurum ATCC 25795
pregna-1,4-diene-3,20-dione + reduced 2,6-dichlorophenol-indophenol
-
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
743221
Mycolicibacterium neoaurum
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
743221
Mycolicibacterium neoaurum NwIB-01
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
androsta-1,4-diene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
743221
Mycolicibacterium neoaurum ATCC 25795
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.3.99.4
?
x * 61000, calculated
Mycolicibacterium neoaurum
1.3.99.4
?
x * 60100, calculated
Mycolicibacterium neoaurum
1.3.99.4
?
x * 54300, calculated
Mycolicibacterium neoaurum
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.14.15.30
30
-
assay at
Mycolicibacterium neoaurum
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.14.15.30
7
-
assay at
Mycolicibacterium neoaurum
Expression
EC Number
Organism
Commentary
Expression
1.14.15.30
Mycolicibacterium neoaurum
recombinant coexpression of the enzyme KshA from Mycobacterium neoaurum strain NwIB-01 and the three homologues of ketosteroid-DELTA1-dehydrogenase (KstD) from Mycobacterium neoaurum ATCC257953 in Escherichia coli strain BL21(DE3),to install a steroid production system with a broad substrate specificity. Enzyme KshA converts the KstD reaction side product 4-androstene-3,17-dione into 9alpha-hydroxyandrosta-1,4-diene-3,17-dione
additional information
Expression (protein specific)
EC Number
Organism
Commentary
Expression
1.14.15.30
Mycolicibacterium neoaurum
recombinant coexpression of the enzyme KshA from Mycobacterium neoaurum strain NwIB-01 and the three homologues of ketosteroid-DELTA1-dehydrogenase (KstD) from Mycobacterium neoaurum ATCC257953 in Escherichia coli strain BL21(DE3),to install a steroid production system with a broad substrate specificity. Enzyme KshA converts the KstD reaction side product 4-androstene-3,17-dione into 9alpha-hydroxyandrosta-1,4-diene-3,17-dione
additional information