BRENDA - Enzyme Database
show all sequences of 1.14.15.30

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)
Commentary
Organism
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
Amino acid exchange
Commentary
Organism
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
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
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
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
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
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
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
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]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
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
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
Metals/Ions
Commentary
Organism
Structure
Fe2+
the enzyme contains a nonheme Fe2+; the enzyme contains a nonheme Fe2+
Rhodococcus rhodochrous
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Rhodococcus rhodochrous
F1CMX0
gene kshA1, encoding an isoform of the oxygenase component of 3-ketosteroid 9alpha-hydroxylase; isozyme KshA1
-
Rhodococcus rhodochrous
F1CMX3
gene kshB, encoding the ferredoxin reductase
-
Rhodococcus rhodochrous
F1CMY8
gene kshA5, encoding an isoform of the oxygenase component of 3-ketosteroid 9alpha-hydroxylase; isozyme KshA5
-
Rhodococcus rhodochrous DSM 43269
F1CMX0
gene kshA1, encoding an isoform of the oxygenase component of 3-ketosteroid 9alpha-hydroxylase; isozyme KshA1
-
Rhodococcus rhodochrous DSM 43269
F1CMX3
gene kshB, encoding the ferredoxin reductase
-
Rhodococcus rhodochrous DSM 43269
F1CMY8
gene kshA5, encoding an isoform of the oxygenase component of 3-ketosteroid 9alpha-hydroxylase; isozyme KshA5
-
Purification (Commentary)
Commentary
Organism
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)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
-
-
-
?
1,4-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
-
-
-
?
1,4-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
-
-
-
?
1,4-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-(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-(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-(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-(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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
?
-
-
-
-
additional information
the KshA5 homologue has a broad substrate range with no apparent preference for any of the tested steroids
717737
Rhodococcus rhodochrous
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at; assay at
Rhodococcus rhodochrous
Cofactor
Cofactor
Commentary
Organism
Structure
additional information
a Rieske nonheme monooxygenase; a Rieske nonheme monooxygenase
Rhodococcus rhodochrous
[2Fe-2S]-center
;
Rhodococcus rhodochrous
Cloned(Commentary) (protein specific)
Commentary
Organism
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
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)
Cofactor
Commentary
Organism
Structure
additional information
a Rieske nonheme monooxygenase
Rhodococcus rhodochrous
[2Fe-2S]-center
-
Rhodococcus rhodochrous
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
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
D230E/S232T/F238Y
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
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
D242W
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
E236D/T238S/Y244F
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
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
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
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
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
Q209T/A210G
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
T207V/T209S/Y211F/H213R/S214G/T215Q/G216A
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
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
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
V201T/S203T/F205Y/R207H/G208S/Q209T/A210G
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
W248D
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
Rhodococcus rhodochrous
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)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
kinetic analysis of the activity of KshA mutants with 4-23,24-bisnorcholesta-4-ene-22-oic acid
Rhodococcus rhodochrous
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
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)
Metals/Ions
Commentary
Organism
Structure
Fe2+
the enzyme contains a nonheme Fe2+
Rhodococcus rhodochrous
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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)
Commentary
Organism
copurification of recombinant His-tagged KshA mutants with His-tagged KshB
Rhodococcus rhodochrous
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
-
-
-
?
1,4-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
-
-
-
?
1,4-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
-
-
-
?
1,4-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-(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-(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-(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-(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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
-
-
-
?
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
?
-
-
-
-
additional information
the KshA5 homologue has a broad substrate range with no apparent preference for any of the tested steroids
717737
Rhodococcus rhodochrous
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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
?
-
-
-
-
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)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Rhodococcus rhodochrous
General Information
General Information
Commentary
Organism
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)
General Information
Commentary
Organism
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
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
Other publictions for EC 1.14.15.30
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
745648
Guevara
Functional characterization o ...
Rhodococcus ruber, Rhodococcus ruber Chol-4
J. Steroid Biochem. Mol. Biol.
172
176-187
2017
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1
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1
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4
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6
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22
1
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1
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1
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4
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22
1
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-
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4
4
-
-
-
746503
Zhang
Efficient 9alpha-hydroxy-4-an ...
Mycolicibacterium neoaurum, Mycolicibacterium neoaurum JC-12
SpringerPlus
5
1207
2016
-
-
1
-
1
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5
-
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8
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2
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1
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1
1
10
2
1
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1
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1
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1
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5
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8
-
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1
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-
1
1
10
2
1
-
-
-
1
-
-
-
-
1
1
-
-
-
743221
Yao
Characterization and engineer ...
Mycolicibacterium neoaurum, Mycolicibacterium neoaurum ATCC 25795, Mycolicibacterium neoaurum NwIB-01
Metab. Eng.
24
181-191
2014
-
-
1
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1
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1
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3
-
4
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3
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1
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1
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1
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1
1
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1
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1
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3
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3
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1
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-
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1
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-
-
1
-
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1
-
-
744058
Petrusma
3-Ketosteroid 9alpha-hydroxyl ...
Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv, Mycolicibacterium smegmatis, Mycolicibacterium smegmatis mc2 155, Rhodococcus erythropolis, Rhodococcus erythropolis SQ1, Rhodococcus jostii, Rhodococcus rhodochrous, Rhodococcus rhodochrous DSM 43269
Antonie van Leeuwenhoek
106
157-172
2014
-
2
2
-
1
-
-
-
-
5
-
22
-
15
-
-
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5
-
-
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28
5
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10
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2
4
14
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1
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7
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22
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28
7
-
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-
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-
-
20
28
-
-
-
745308
Penfield
Substrate specificities and c ...
Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv, Rhodococcus rhodochrous, Rhodococcus rhodochrous DSM43269, Rhodococcus rhodochrous DSM 43269
J. Biol. Chem.
289
25523-25536
2014
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1
2
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4
19
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8
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18
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1
2
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54
2
2
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19
2
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3
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2
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3
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6
3
20
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8
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2
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54
3
3
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19
3
-
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4
6
-
17
17
745698
Yeh
Deletion of the gene encoding ...
Rhodococcus hoagii 103S, Rhodococcus hoagii
Microb. Cell Fact.
13
130
2014
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1
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1
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4
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2
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4
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1
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1
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1
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4
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4
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1
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1
1
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-
-
717737
Petrusma
Structural features in the Ksh ...
Rhodococcus rhodochrous, Rhodococcus rhodochrous DSM 43269
J. Bacteriol.
194
115-121
2012
-
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1
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8
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2
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1
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3
-
13
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1
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74
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1
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2
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3
4
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16
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4
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2
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3
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3
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74
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2
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1
2
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-
717735
Petrusma
Multiplicity of 3-ketosteroid- ...
Rhodococcus rhodochrous, Rhodococcus rhodochrous DSM 43269
J. Bacteriol.
193
3931-3940
2011
-
-
1
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1
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4
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19
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1
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1
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75
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1
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1
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6
5
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5
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4
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6
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3
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75
-
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3
-
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1
3
11
5
-
-
717854
Capyk
Activity of 3-ketosteroid 9alp ...
Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv
J. Biol. Chem.
286
40717-40724
2011
-
-
-
-
-
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13
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2
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9
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30
1
1
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11
1
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1
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1
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21
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2
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30
2
2
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19
2
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2
3
-
13
19
713784
Wei
A new steroid-transforming str ...
Mycolicibacterium neoaurum, Mycolicibacterium neoaurum NwIB-01
Appl. Biochem. Biotechnol.
162
1446-1456
2010
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1
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3
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1
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1
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1
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2
1
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1
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1
1
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716250
Hu
3-Ketosteroid 9alpha-hydroxyla ...
Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv
Mol. Microbiol.
75
107-121
2010
-
-
-
-
-
-
-
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160
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4
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4
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2
2
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717500
Fan
Cloning, heterologous expressi ...
Mycobacterium sp., Mycobacterium sp. NwIB-01
Chin. J. Biotechnol.
25
2014-2021
2009
-
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1
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1
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8
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1
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1
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1
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1
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1
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1
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717784
Capyk
Characterization of 3-ketoster ...
Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv
J. Biol. Chem.
284
9937-9946
2009
-
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1
1
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3
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6
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1
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4
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3
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1
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1
1
1
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3
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1
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4
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3
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3
3
717119
Van Der Geize
Characterization of a second R ...
Rhodococcus erythropolis, Rhodococcus erythropolis SQ1
Appl. Environ. Microbiol.
74
7197-7203
2008
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9
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8
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8
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1
2
3
1
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717118
Andor
Generation of useful insertion ...
Mycolicibacterium smegmatis, Mycolicibacterium smegmatis mc(2)155 / ATCC 700084
Appl. Environ. Microbiol.
72
6554-6559
2006
-
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1
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1
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1
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718125
Van Der Geize
Molecular and functional chara ...
Rhodococcus erythropolis, Rhodococcus erythropolis SQ1
Mol. Microbiol.
45
1007-1018
2002
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1
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1
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4
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8
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1
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2
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8
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