BRENDA - Enzyme Database

Structural features in the KshA terminal oxygenase protein that determine substrate preference of 3-ketosteroid 9alpha-hydroxylase enzymes

Petrusma, M.; Dijkhuizen, L.; van Der Geize, R.; J. Bacteriol. 194, 115-121 (2012)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.14.15.30
expressed in Escherichia coli C41(DE3) cells; expressed in Escherichia coli C41(DE3) cells; expression of chimeric kshA mutant genes, KshA1A5beta and KshA5A1beta, in Escherichia coli, coexpression of the His-tagged KshA mutants with His-tagged KshB, molar ratio of KshA5A1/KshB is 1:8, low expression level of soluble KshA5A1alpha enzyme; expression of chimeric kshA mutant genes, KshA1A5beta and KshA5A1beta, in Escherichia coli, coexpression of the His-tagged KshA mutants with His-tagged KshB, molar ratio of KshA5A1/KshB is 1:8, low expression level of soluble KshA5A1alpha enzyme; expression of chimeric kshA mutant genes, KshA1A5beta and KshA5A1beta, in Escherichia coli, coexpression of the His-tagged KshA mutants with His-tagged KshB, molar ratio of KshA5A1/KshB is 1:8, low expression level of soluble KshA5A1alpha enzyme
Rhodococcus rhodochrous
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.14.15.30
D230E/S232T/F238Y
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA1 shows no 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
D242W
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA1 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
E236D/T238S/Y244F
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA5 shows no 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
additional information
construction of chimeric KshA enzyme mutants with exchanged helix-grip fold beta-sheets that retain KSH activity. kshA1 E198 to V255 is exchanged for kshA5 E204 to I261. kshA1 I257 to I306 is exchanged for kshA5 I263 to V312, kshA5 E204 to I261 is exchanged for kshA1 E198 to V255, kshA5 I263 to V312 is exchanged for kshA1 I257 to I306, kshA1 209-QAREDTRPHANGQPKMIGS-227is exchanged for kshA5 215-TGREDVISGTNYDDPNAEL-233, and kshA5 215-TGREDVISGTNYDDPNAEL-233 is exchanged for kshA1 209-QAREDTRPHANGQPKMIGS-227; construction of chimeric KshA enzyme mutants with exchanged helix-grip fold beta-sheets that retain KSH activity. kshA1 E198 to V255 is exchanged for kshA5 E204 to I261. kshA1 I257 to I306 is exchanged for kshA5 I263 to V312, kshA5 E204 to I261 is exchanged for kshA1 E198 to V255, kshA5 I263 to V312 is exchanged for kshA1 I257 to I306, kshA1 209-QAREDTRPHANGQPKMIGS-227is exchanged for kshA5 215-TGREDVISGTNYDDPNAEL-233, and kshA5 215-TGREDVISGTNYDDPNAEL-233 is exchanged for kshA1 209-QAREDTRPHANGQPKMIGS-227; mutant KshA1A5beta is isoform KshA1 with beta sheet of KshA5 and the mutations kshA1 E198 to V255 exchanged for kshA5 E204 to I261, and exhibits 3-ketosteroid 9alpha-hydroxylase activity. Mutant KshA1A5alpha is isoform KshA1 with alpha helix of KshA5 and mutations kshA1 I257 to I306 exchanged for kshA5 I263 to V312, and shows not enzymatic activity. Mutant KshA1A5loop is isoform KshA1 with loop region of KshA5 and mutation kshA1 209-QAREDTRPHANGQPKMIGS-227 exchanged for kshA5 215-TGREDVISGTNYDDPNAEL-233, and exhibits 3-ketosteroid 9alpha-hydroxylase activity; mutant KshA5A1beta is isoform KshA5 with beta sheet of KshA1 and mutation kshA5 E204 to I261 exchanged for kshA1 E198 to V255, and exhibits 3-ketosteroid 9alpha-hydroxylase activity. Mutant KshA5A1alpha is isoform KshA5 with alpha helix of KshA1 and mutation kshA5 I263 to V312 exchanged for kshA1 I257 to I306, and shows initial 3-ketosteroid 9alpha-hydroxylase activity. Mutant KshA5A1loop is isoform KshA5 with loop region of KshA1 and mutation kshA5 215-TGREDVISGTNYDDPNAEL-233 exchanged for kshA1 209-QAREDTRPHANGQPKMIGS-227, and exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
Q209T/A210G
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA1 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
T207V/T209S/Y211F/H213R/S214G/T215Q/G216A
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA5 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
V201T/S203T/F205Y/R207H/G208S/Q209T/A210G
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA1 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
W248D
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA5 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.15.30
additional information
-
additional information
kinetic analysis of the activity of KshA mutants with 4-23,24-bisnorcholesta-4-ene-22-oic acid; kinetic analysis of the activity of KshA mutants with 4-23,24-bisnorcholesta-4-ene-22-oic acid
Rhodococcus rhodochrous
1.14.15.30
0.01
-
23,24-bisnorcholesta-4-ene-22-oic acid
Km less than 0.01 mM, isoform KshA1, in 50mMTris-HCl buffer (pH 7.0), temperature not specified in the publication; Km less than 0.01 mM, isoform KshA5, in 50 mM Tris-HCl buffer (pH 7.0), temperature not specified in the publication
Rhodococcus rhodochrous
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.14.15.30
Fe2+
the enzyme contains a nonheme Fe2+; the enzyme contains a nonheme Fe2+
Rhodococcus rhodochrous
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
Rhodococcus rhodochrous
-
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
1.14.15.30
Rhodococcus rhodochrous
F1CMX0
gene kshA1, encoding an isoform of the oxygenase component of 3-ketosteroid 9alpha-hydroxylase; isozyme KshA1
-
1.14.15.30
Rhodococcus rhodochrous
F1CMX3
gene kshB, encoding the ferredoxin reductase
-
1.14.15.30
Rhodococcus rhodochrous
F1CMY8
gene kshA5, encoding an isoform of the oxygenase component of 3-ketosteroid 9alpha-hydroxylase; isozyme KshA5
-
1.14.15.30
Rhodococcus rhodochrous DSM 43269
F1CMX0
gene kshA1, encoding an isoform of the oxygenase component of 3-ketosteroid 9alpha-hydroxylase; isozyme KshA1
-
1.14.15.30
Rhodococcus rhodochrous DSM 43269
F1CMX3
gene kshB, encoding the ferredoxin reductase
-
1.14.15.30
Rhodococcus rhodochrous DSM 43269
F1CMY8
gene kshA5, encoding an isoform of the oxygenase component of 3-ketosteroid 9alpha-hydroxylase; isozyme KshA5
-
Purification (Commentary)
EC Number
Commentary
Organism
1.14.15.30
copurification of recombinant His-tagged KshA mutants with His-tagged KshB; copurification of recombinant His-tagged KshA mutants with His-tagged KshB; copurification of recombinant His-tagged KshA mutants with His-tagged KshB
Rhodococcus rhodochrous
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
the wild type homologue KshA1 shows 244% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
? + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 51% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
? + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 244% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous DSM 43269
? + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 51% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous DSM 43269
? + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1-(5alpha)-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androst-1-en-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1-(5alpha)-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 23% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androst-1-en-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1-(5alpha)-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 99% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androst-1-en-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1-(5alpha)-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha-hydroxy-5alpha-androst-1-en-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
11beta-hydrocortisone + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-11beta-hydrocortisone + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
11beta-hydrocortisone + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 93% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-11beta-hydrocortisone + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
11beta-hydrocortisone + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha-hydroxy-11beta-hydrocortisone + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
19-nor-4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-19-nor-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
19-nor-4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. nordion, the wild type homologue KshA1 shows 24% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-19-nor-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
19-nor-4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. nordion, the wild type homologue KshA5 shows 111% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-19-nor-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
19-nor-4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha-hydroxy-19-nor-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
23,24-bisnorcholest-4-en-22-oic acid + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 548% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-23,24-bisnorcholest-4-en-22-oic acid + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
23,24-bisnorcholest-4-en-22-oic acid + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 88% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-23,24-bisnorcholest-4-en-22-oic acid + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
3-oxo-23,24-bisnorcholest-4-en-22-oic acid + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-3-oxo-23,24-bisnorcholest-4-en-22-oic acid + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
3-oxo-23,24-bisnorcholest-4-en-22-oic acid + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha-hydroxy-3-oxo-23,24-bisnorcholest-4-en-22-oic acid + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-4-androstene-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. testosterone, the wild type homologue KshA1 shows 158% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-4-androstene-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. testosterone, the wild type homologue KshA5 shows 113% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-4-androstene-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha,17beta-dihydroxy-4-androstene-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 100% activity
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 100% activity
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-pregnene-3,20-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-pregnene-3,20-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-pregnene-3,20-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. progesterone, the wild type homologue KshA1 shows 372% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-pregnene-3,20-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-pregnene-3,20-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. progesterone, the wild type homologue KshA5 shows 65% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-pregnene-3,20-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-5alpha-androstane-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. stanolon, the wild type homologue KshA5 shows 100% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-5alpha-androstane-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 12% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 73% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5beta-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5beta-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5beta-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 19% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5beta-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5beta-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 99% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5beta-androstane-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
-
717737
Rhodococcus rhodochrous
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
additional information
the KshA1 homologue has a narrow substrate range with a high preference for 23,24-bisnorcholesta-4-ene-22-oic acid and 23,24-bisnorcholesta-1,4-diene-22-oic acid and preference to a lesser extent for 1,4-androstadiene-3,17-dione and 4-pregnene-3,20-dione (progesterone)
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
the KshA5 homologue has a broad substrate range with no apparent preference for any of the tested steroids
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
the wild type homologue KshA1 shows no activity with 5alpha-androstane-17beta-ol-3-one (stanolon), 5-cholestene-3beta-ol (cholesterol), 3alpha-hydroxy-5alpha-pregnane-20-one, 11beta-hydrocortisone, 3beta-hydroxy-5alpha-androstane-17-one, and 9alpha-hydroxy-4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
the wild type homologue KshA5 shows no activity with 5-cholestene-3beta-ol, 3alpha-hydroxy-5beta-pregnane-20-one, 3beta-hydroxy-5alpha-androstane-17-one, and 9alpha-hydroxy-4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
substrate specificities of KshA variants, overview. No activity by all enzymes with 3alpha-hydroxy-5alpha-pregnane-20-one, 9alpha-hydroxy-4-androstene-3,17-dione, 5-cholestene-3beta-ol, and 3beta-hydroxy-5alpha-androstane-17-one
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
the KshA1 homologue has a narrow substrate range with a high preference for 23,24-bisnorcholesta-4-ene-22-oic acid and 23,24-bisnorcholesta-1,4-diene-22-oic acid and preference to a lesser extent for 1,4-androstadiene-3,17-dione and 4-pregnene-3,20-dione (progesterone)
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
1.14.15.30
additional information
the wild type homologue KshA1 shows no activity with 5alpha-androstane-17beta-ol-3-one (stanolon), 5-cholestene-3beta-ol (cholesterol), 3alpha-hydroxy-5alpha-pregnane-20-one, 11beta-hydrocortisone, 3beta-hydroxy-5alpha-androstane-17-one, and 9alpha-hydroxy-4-androstene-3,17-dione
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
1.14.15.30
additional information
substrate specificities of KshA variants, overview. No activity by all enzymes with 3alpha-hydroxy-5alpha-pregnane-20-one, 9alpha-hydroxy-4-androstene-3,17-dione, 5-cholestene-3beta-ol, and 3beta-hydroxy-5alpha-androstane-17-one
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
1.14.15.30
additional information
the KshA5 homologue has a broad substrate range with no apparent preference for any of the tested steroids
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
1.14.15.30
additional information
the wild type homologue KshA5 shows no activity with 5-cholestene-3beta-ol, 3alpha-hydroxy-5beta-pregnane-20-one, 3beta-hydroxy-5alpha-androstane-17-one, and 9alpha-hydroxy-4-androstene-3,17-dione
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.14.15.30
7
-
assay at; assay at
Rhodococcus rhodochrous
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.14.15.30
additional information
a Rieske nonheme monooxygenase; a Rieske nonheme monooxygenase
Rhodococcus rhodochrous
1.14.15.30
[2Fe-2S]-center
;
Rhodococcus rhodochrous
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.14.15.30
expressed in Escherichia coli C41(DE3) cells; expression of chimeric kshA mutant genes, KshA1A5beta and KshA5A1beta, in Escherichia coli, coexpression of the His-tagged KshA mutants with His-tagged KshB, molar ratio of KshA5A1/KshB is 1:8, low expression level of soluble KshA5A1alpha enzyme
Rhodococcus rhodochrous
1.14.15.30
expression of chimeric kshA mutant genes, KshA1A5beta and KshA5A1beta, in Escherichia coli, coexpression of the His-tagged KshA mutants with His-tagged KshB, molar ratio of KshA5A1/KshB is 1:8, low expression level of soluble KshA5A1alpha enzyme
Rhodococcus rhodochrous
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.14.15.30
additional information
a Rieske nonheme monooxygenase
Rhodococcus rhodochrous
1.14.15.30
[2Fe-2S]-center
-
Rhodococcus rhodochrous
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.14.15.30
D230E/S232T/F238Y
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA1 shows no 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
D230E/S232T/F238Y
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
1.14.15.30
D242W
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA1 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
D242W
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
1.14.15.30
E236D/T238S/Y244F
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
1.14.15.30
E236D/T238S/Y244F
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA5 shows no 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
additional information
construction of chimeric KshA enzyme mutants with exchanged helix-grip fold beta-sheets that retain KSH activity. kshA1 E198 to V255 is exchanged for kshA5 E204 to I261. kshA1 I257 to I306 is exchanged for kshA5 I263 to V312, kshA5 E204 to I261 is exchanged for kshA1 E198 to V255, kshA5 I263 to V312 is exchanged for kshA1 I257 to I306, kshA1 209-QAREDTRPHANGQPKMIGS-227is exchanged for kshA5 215-TGREDVISGTNYDDPNAEL-233, and kshA5 215-TGREDVISGTNYDDPNAEL-233 is exchanged for kshA1 209-QAREDTRPHANGQPKMIGS-227; mutant KshA1A5beta is isoform KshA1 with beta sheet of KshA5 and the mutations kshA1 E198 to V255 exchanged for kshA5 E204 to I261, and exhibits 3-ketosteroid 9alpha-hydroxylase activity. Mutant KshA1A5alpha is isoform KshA1 with alpha helix of KshA5 and mutations kshA1 I257 to I306 exchanged for kshA5 I263 to V312, and shows not enzymatic activity. Mutant KshA1A5loop is isoform KshA1 with loop region of KshA5 and mutation kshA1 209-QAREDTRPHANGQPKMIGS-227 exchanged for kshA5 215-TGREDVISGTNYDDPNAEL-233, and exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
additional information
construction of chimeric KshA enzyme mutants with exchanged helix-grip fold beta-sheets that retain KSH activity. kshA1 E198 to V255 is exchanged for kshA5 E204 to I261. kshA1 I257 to I306 is exchanged for kshA5 I263 to V312, kshA5 E204 to I261 is exchanged for kshA1 E198 to V255, kshA5 I263 to V312 is exchanged for kshA1 I257 to I306, kshA1 209-QAREDTRPHANGQPKMIGS-227is exchanged for kshA5 215-TGREDVISGTNYDDPNAEL-233, and kshA5 215-TGREDVISGTNYDDPNAEL-233 is exchanged for kshA1 209-QAREDTRPHANGQPKMIGS-227; mutant KshA5A1beta is isoform KshA5 with beta sheet of KshA1 and mutation kshA5 E204 to I261 exchanged for kshA1 E198 to V255, and exhibits 3-ketosteroid 9alpha-hydroxylase activity. Mutant KshA5A1alpha is isoform KshA5 with alpha helix of KshA1 and mutation kshA5 I263 to V312 exchanged for kshA1 I257 to I306, and shows initial 3-ketosteroid 9alpha-hydroxylase activity. Mutant KshA5A1loop is isoform KshA5 with loop region of KshA1 and mutation kshA5 215-TGREDVISGTNYDDPNAEL-233 exchanged for kshA1 209-QAREDTRPHANGQPKMIGS-227, and exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
Q209T/A210G
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA1 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
Q209T/A210G
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
1.14.15.30
T207V/T209S/Y211F/H213R/S214G/T215Q/G216A
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
1.14.15.30
T207V/T209S/Y211F/H213R/S214G/T215Q/G216A
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA5 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
V201T/S203T/F205Y/R207H/G208S/Q209T/A210G
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA1 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
1.14.15.30
V201T/S203T/F205Y/R207H/G208S/Q209T/A210G
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
1.14.15.30
W248D
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
1.14.15.30
W248D
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme; the mutant of isoform KshA5 exhibits 3-ketosteroid 9alpha-hydroxylase activity
Rhodococcus rhodochrous
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.15.30
additional information
-
additional information
kinetic analysis of the activity of KshA mutants with 4-23,24-bisnorcholesta-4-ene-22-oic acid
Rhodococcus rhodochrous
1.14.15.30
0.01
-
23,24-bisnorcholesta-4-ene-22-oic acid
Km less than 0.01 mM, isoform KshA1, in 50mMTris-HCl buffer (pH 7.0), temperature not specified in the publication
Rhodococcus rhodochrous
1.14.15.30
0.01
-
23,24-bisnorcholesta-4-ene-22-oic acid
Km less than 0.01 mM, isoform KshA5, in 50 mM Tris-HCl buffer (pH 7.0), temperature not specified in the publication
Rhodococcus rhodochrous
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.14.15.30
Fe2+
the enzyme contains a nonheme Fe2+
Rhodococcus rhodochrous
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
Rhodococcus rhodochrous
-
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.14.15.30
copurification of recombinant His-tagged KshA mutants with His-tagged KshB
Rhodococcus rhodochrous
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
the wild type homologue KshA1 shows 244% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
? + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 51% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
? + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 244% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous DSM 43269
? + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1,4-androstadiene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 51% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous DSM 43269
? + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1-(5alpha)-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androst-1-en-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1-(5alpha)-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 23% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androst-1-en-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1-(5alpha)-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 99% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androst-1-en-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
1-(5alpha)-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha-hydroxy-5alpha-androst-1-en-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
11beta-hydrocortisone + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-11beta-hydrocortisone + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
11beta-hydrocortisone + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 93% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-11beta-hydrocortisone + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
11beta-hydrocortisone + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha-hydroxy-11beta-hydrocortisone + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
19-nor-4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-19-nor-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
19-nor-4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. nordion, the wild type homologue KshA1 shows 24% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-19-nor-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
19-nor-4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. nordion, the wild type homologue KshA5 shows 111% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-19-nor-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
19-nor-4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha-hydroxy-19-nor-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
23,24-bisnorcholest-4-en-22-oic acid + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 548% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-23,24-bisnorcholest-4-en-22-oic acid + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
23,24-bisnorcholest-4-en-22-oic acid + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 88% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-23,24-bisnorcholest-4-en-22-oic acid + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
3-oxo-23,24-bisnorcholest-4-en-22-oic acid + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-3-oxo-23,24-bisnorcholest-4-en-22-oic acid + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
3-oxo-23,24-bisnorcholest-4-en-22-oic acid + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha-hydroxy-3-oxo-23,24-bisnorcholest-4-en-22-oic acid + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-4-androstene-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. testosterone, the wild type homologue KshA1 shows 158% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-4-androstene-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. testosterone, the wild type homologue KshA5 shows 113% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-4-androstene-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous DSM 43269
9alpha,17beta-dihydroxy-4-androstene-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 100% activity
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-androstene-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 100% activity
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-androstene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-pregnene-3,20-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-pregnene-3,20-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-pregnene-3,20-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. progesterone, the wild type homologue KshA1 shows 372% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-pregnene-3,20-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
4-pregnene-3,20-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. progesterone, the wild type homologue KshA5 shows 65% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-4-pregnene-3,20-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-5alpha-androstane-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-17beta-ol-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
i.e. stanolon, the wild type homologue KshA5 shows 100% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha,17beta-dihydroxy-5alpha-androstane-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 12% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5alpha-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 73% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5alpha-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5beta-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
-
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5beta-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5beta-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA1 shows 19% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5beta-androstane-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
5beta-androstane-3,17-dione + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
the wild type homologue KshA5 shows 99% activity compared to 4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
9alpha-hydroxy-5beta-androstane-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
-
717737
Rhodococcus rhodochrous
9alpha-hydroxyandrosta-1,4-diene-3,17-dione + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
-
-
-
?
1.14.15.30
additional information
the KshA1 homologue has a narrow substrate range with a high preference for 23,24-bisnorcholesta-4-ene-22-oic acid and 23,24-bisnorcholesta-1,4-diene-22-oic acid and preference to a lesser extent for 1,4-androstadiene-3,17-dione and 4-pregnene-3,20-dione (progesterone)
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
the KshA5 homologue has a broad substrate range with no apparent preference for any of the tested steroids
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
the wild type homologue KshA1 shows no activity with 5alpha-androstane-17beta-ol-3-one (stanolon), 5-cholestene-3beta-ol (cholesterol), 3alpha-hydroxy-5alpha-pregnane-20-one, 11beta-hydrocortisone, 3beta-hydroxy-5alpha-androstane-17-one, and 9alpha-hydroxy-4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
the wild type homologue KshA5 shows no activity with 5-cholestene-3beta-ol, 3alpha-hydroxy-5beta-pregnane-20-one, 3beta-hydroxy-5alpha-androstane-17-one, and 9alpha-hydroxy-4-androstene-3,17-dione
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
substrate specificities of KshA variants, overview. No activity by all enzymes with 3alpha-hydroxy-5alpha-pregnane-20-one, 9alpha-hydroxy-4-androstene-3,17-dione, 5-cholestene-3beta-ol, and 3beta-hydroxy-5alpha-androstane-17-one
717737
Rhodococcus rhodochrous
?
-
-
-
-
1.14.15.30
additional information
the KshA1 homologue has a narrow substrate range with a high preference for 23,24-bisnorcholesta-4-ene-22-oic acid and 23,24-bisnorcholesta-1,4-diene-22-oic acid and preference to a lesser extent for 1,4-androstadiene-3,17-dione and 4-pregnene-3,20-dione (progesterone)
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
1.14.15.30
additional information
the wild type homologue KshA1 shows no activity with 5alpha-androstane-17beta-ol-3-one (stanolon), 5-cholestene-3beta-ol (cholesterol), 3alpha-hydroxy-5alpha-pregnane-20-one, 11beta-hydrocortisone, 3beta-hydroxy-5alpha-androstane-17-one, and 9alpha-hydroxy-4-androstene-3,17-dione
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
1.14.15.30
additional information
substrate specificities of KshA variants, overview. No activity by all enzymes with 3alpha-hydroxy-5alpha-pregnane-20-one, 9alpha-hydroxy-4-androstene-3,17-dione, 5-cholestene-3beta-ol, and 3beta-hydroxy-5alpha-androstane-17-one
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
1.14.15.30
additional information
the KshA5 homologue has a broad substrate range with no apparent preference for any of the tested steroids
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
1.14.15.30
additional information
the wild type homologue KshA5 shows no activity with 5-cholestene-3beta-ol, 3alpha-hydroxy-5beta-pregnane-20-one, 3beta-hydroxy-5alpha-androstane-17-one, and 9alpha-hydroxy-4-androstene-3,17-dione
717737
Rhodococcus rhodochrous DSM 43269
?
-
-
-
-
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.14.15.30
7
-
assay at
Rhodococcus rhodochrous
General Information
EC Number
General Information
Commentary
Organism
1.14.15.30
additional information
determination of the enzyme's substrate preference, three-dimensional structure modeling of KshA1, overview. The variable loop within the beta-sheet plays a role in the substrate preference of KshA enzymes; determination of the enzyme's substrate preference, three-dimensional structure modeling of KshA5, overview. The variable loop within the beta-sheet plays a role in the substrate preference of KshA enzymes
Rhodococcus rhodochrous
General Information (protein specific)
EC Number
General Information
Commentary
Organism
1.14.15.30
additional information
determination of the enzyme's substrate preference, three-dimensional structure modeling of KshA1, overview. The variable loop within the beta-sheet plays a role in the substrate preference of KshA enzymes
Rhodococcus rhodochrous
1.14.15.30
additional information
determination of the enzyme's substrate preference, three-dimensional structure modeling of KshA5, overview. The variable loop within the beta-sheet plays a role in the substrate preference of KshA enzymes
Rhodococcus rhodochrous