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Enzyme Nomenclature
Information on EC 1.14.15.8 - steroid 15beta-monooxygenase
Word Map on EC 1.14.15.8
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The expected taxonomic range for this enzyme is: Bacillus megaterium
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EC NUMBER 
COMMENTARY 
1.14.15.8
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RECOMMENDED NAME
GeneOntology No.
steroid 15beta-monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
progesterone + 2 reduced [2Fe-2S] ferredoxin + O2 = 15beta-hydroxyprogesterone + 2 oxidized [2Fe-2S] ferredoxin + H2O
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SYSTEMATIC NAME
IUBMB Comments
progesterone,reduced-ferredoxin:oxygen oxidoreductase (15beta-hydroxylating)
The enzyme from the bacterium Bacillus megaterium hydroxylates a variety of 3-oxo-Delta4-steroids in position 15beta. Ring A-reduced, aromatic, and 3beta-hydroxy-Delta4-steroids do not serve as substrates [2].
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ATCC 13368
SwissProt
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
11-deoxycorticosterone + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized adrenodoxin + H2O
11-deoxycorticosterone + reduced ferredoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized ferredoxin + H2O
ferredoxin from the cyanobacterium Anabaena PCC 7119
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-
?
11-deoxycorticosterone + reduced flavodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized flavodoxin + H2O
flavodoxin from the cyanobacterium Anabaena PCC 7119
-
-
?
11-deoxycorticosterone + reduced megaredoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized megaredoxin + H2O
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megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
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-
?
11-deoxycortisol + reduced adrenodoxin + O2
15beta-15,17,21-trihydroxypregn-4-ene-3,20-dione + oxidized adrenodoxin + H2O
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adrenodoxin is a [2Fe-2S] ferredoxin involved in electron transfer from NADPH+ (via a reductase) to cytochrome P-450 in the adrenal gland
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-
?
11-oxo-beta-boswellic acid + reduced ferredoxin + O2
(3alpha,15beta)-3,15-dihydroxy-11-oxours-12-en-24-oic acid + H2O
17alpha-hydroxyprogesterone + reduced megaredoxin + O2
(15beta,17alpha)-15,17-dihydroxyprogesterone + oxidized megaredoxin + H2O
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megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
-
-
?
20alpha-dihydroprogesterone + reduced megaredoxin + O2
(15beta,20alpha)-15-hydroxy-20-dihydroprogesterone + oxidized megaredoxin + H2O
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megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
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-
?
4-androstene-3,17-dione + reduced megaredoxin + O2
15beta-hydroxyandrostene-3,17-dione + oxidized megaredoxin + H2O
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megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
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-
?
6alpha-fluoro-16alpha-methyl-deoxycorticosterone + reduced ferredoxin + O2
15beta-hydroxy-6alpha-fluoro-16alpha-methyl-deoxycorticosterone + oxidized ferredoxin + H2O
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-
-
-
?
abietic acid + reduced adrenodoxin + O2
12-hydroxyabietic acid + oxidized adrenodoxin + H2O
-
-
-
-
?
corticosterone + reduced megaredoxin + O2
15beta-hydroxycorticosterone + oxidized megaredoxin + H2O
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megaredoxin is an iron-sulfur protein of Bacillus megaterium, preferred substrate, megaredoxin has an apparent sulfur to iron ratio of 0.98
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-
?
imipramine + reduced adrenodoxin + O2
desipramine + oxidized adrenodoxin + H2O
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-
-
-
?
progesterone + reduced adrenodoxin + O2
15beta-hydroxy-progesterone + oxidized adrenodoxin + H2O
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adrenodoxin is a [2Fe-2S] ferredoxin involved in electron transfer from NADPH+ (via a reductase) to cytochrome P-450 in the adrenal gland, 15beta-hydroxy-progesterone is the main product of wild-type enzyme and all mutants. In order to gain insights into the structure and function of CYP106A2, whose crystal structure is unknown, a homology model has been created. The substrate progesterone is then docked into the active site to predict which residues might affect substrate binding. The model is substantiated by using a combination of theoretical and experimental investigations
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?
progesterone + reduced adrenodoxin + O2
15beta-hydroxyprogesterone + oxidized adrenodoxin + H2O
progesterone + reduced ferredoxin + O2
15beta-hydroxy-progesterone + oxidized ferredoxin + H2O
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Bacillus megaterium ferredoxin may be replaced by adrenal ferredoxin
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-
?
progesterone + reduced megaredoxin + O2
15beta-hydroxyprogesterone + oxidized megaredoxin + H2O
testosterone + reduced acceptor + O2
15beta-hydroxytestosterone + oxidized acceptor + H2O
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the major product is identified as 15beta-hydroxytestosterone. 6beta-Hydroxytestosterone and androst-4-ene-3,17-dione are present as minor products
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-
?
testosterone + reduced adrenodoxin + O2
15beta-hydroxytestosterone + oxidized adrenodoxin + H2O
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-
-
-
?
testosterone + reduced megaredoxin + O2
15beta-hydroxytestosterone + oxidized megaredoxin + H2O
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megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
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-
?
11-deoxycorticosterone + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized adrenodoxin + H2O
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-
-
-
?
11-deoxycorticosterone + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized adrenodoxin + H2O
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adrenodoxin is a [2Fe-2S] ferredoxin involved in electron transfer from NADPH+ (via a reductase) to cytochrome P-450 in the adrenal gland, 15beta-hydroxy-deoxycorticosterone is the only product that is observed, both in the case of wild-type CYP106A2 and all the mutants (except T396R)
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-
?
11-deoxycorticosterone + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized adrenodoxin + H2O
deoxycorticosterone binds in the heme pocket near the iron ligand
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-
?
11-oxo-beta-boswellic acid + reduced ferredoxin + O2
(3alpha,15beta)-3,15-dihydroxy-11-oxours-12-en-24-oic acid + H2O
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a pentacyclic triterpene, 15beta-hydroxylation
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-
?
11-oxo-beta-boswellic acid + reduced ferredoxin + O2
(3alpha,15beta)-3,15-dihydroxy-11-oxours-12-en-24-oic acid + H2O
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a pentacyclic triterpene, 15beta-hydroxylation
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-
?
abietic acid + reduced ferredoxin + O2
?
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a pentacyclic triterpene
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-
?
abietic acid + reduced ferredoxin + O2
?
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a pentacyclic triterpene
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-
?
progesterone + reduced adrenodoxin + O2
15beta-hydroxyprogesterone + oxidized adrenodoxin + H2O
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adrenodoxin is a [2Fe-2S] ferredoxin involved in electron transfer from NADPH+ (via a reductase) to cytochrome P-450 in the adrenal gland
-
-
?
progesterone + reduced adrenodoxin + O2
15beta-hydroxyprogesterone + oxidized adrenodoxin + H2O
identification of monohydroxy progesterones using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry. 15beta-Hydroxy-progesterone is the main product. 6beta-Hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone are formed at small amounts
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-
?
progesterone + reduced megaredoxin + O2
15beta-hydroxyprogesterone + oxidized megaredoxin + H2O
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-
-
-
?
progesterone + reduced megaredoxin + O2
15beta-hydroxyprogesterone + oxidized megaredoxin + H2O
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megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
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-
?
additional information
?
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hydroxylates a variety of 3-oxo-DELTA4-steroids in position 15beta in the presence of NADPH and O2. Ring A-reduced, aromatic, and 3beta-hydroxy-DELTA4-steroids do not serve as substrates
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additional information
?
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the specificity of hydroxylation in beta-position can be altered by the choice of the electron transfer system. Replacing the natural electron transfer partners by peroxides, the ratio of 15alpha-/15beta-hydroxylation of progesterone is increased 1.3fold
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additional information
?
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CYP106A2 catalyzes hydroxylations of a variety of 3-oxo-D4-steroids such as progesterone and deoxycorticosterone, mainly in the 15beta-position. The diterpene resin acid abietic acid is a substrate, that is converted to 12alpha- and 12beta-hydroxyabietic acid, catalytic site binding structure, homology modelling, overview
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additional information
?
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high-throughput substrate screening and binding affinities, substrate docking, overview
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additional information
?
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CYP106A2 from hydroxylates a variety of 3-oxo-DELTA4 steroids and catalyzes a one-step regioselective allylic hydroxylation of the diterpene abietic acid
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additional information
?
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enzyme reaction is coupled to NADPH oxidation, overview
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additional information
?
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CYP106A2 from hydroxylates a variety of 3-oxo-DELTA4 steroids and catalyzes a one-step regioselective allylic hydroxylation of the diterpene abietic acid
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-
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additional information
?
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CYP106A2 catalyzes hydroxylations of a variety of 3-oxo-D4-steroids such as progesterone and deoxycorticosterone, mainly in the 15beta-position. The diterpene resin acid abietic acid is a substrate, that is converted to 12alpha- and 12beta-hydroxyabietic acid, catalytic site binding structure, homology modelling, overview
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-
-
additional information
?
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high-throughput substrate screening and binding affinities, substrate docking, overview
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
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Q06069
CYP106A2 catalyzes hydroxylations of a variety of 3-oxo-D4-steroids such as progesterone and deoxycorticosterone, mainly in the 15beta-position. The diterpene resin acid abietic acid is a substrate, that is converted to 12alpha- and 12beta-hydroxyabietic acid, catalytic site binding structure, homology modelling, overview
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-
-
additional information
?
-
Q06069
CYP106A2 catalyzes hydroxylations of a variety of 3-oxo-D4-steroids such as progesterone and deoxycorticosterone, mainly in the 15beta-position. The diterpene resin acid abietic acid is a substrate, that is converted to 12alpha- and 12beta-hydroxyabietic acid, catalytic site binding structure, homology modelling, overview
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cytochrome P450meg
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it is possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing flavoprotein (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system is fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin has an apparent sulfur to iron ratio of 0.98 and shows g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic resonance spectroscopy
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heme
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the heme content of cytochrome P-450meg is 0.94 nmol of heme per nmol of cytochrome P-450
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
heme
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the heme content of cytochrome P-450meg is 0.94 nmol of heme per nmol of cytochrome P-450
Iron
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it is possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing flavoprotein (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system is fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin has an apparent sulfur to iron ratio of 0.98 and shows g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic resonance spectroscopy
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00314
Ferredoxin
pH 7.4, 30°C, wild-type ferredoxin from the cyanobacterium Anabaena PCC 7119
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0.00734
flavodoxin
pH 7.4, 30°C, wild-type flavodoxin from the cyanobacterium Anabaena PCC 7119
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additional information
additional information
KM-value for the deoxicorticosterone 15beta-hydroxylase activity of recombinant BmCYP106A2 when using the NADPH/Anabaena ferredoxin electron donor system and different electron carrier proteins. Use of different AnFld mutants at positions W57, I59 and I92, which alter their aromatic and hydrophobic character, as electron donor to BmCYP106A2
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0.037
11-deoxycortisol
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pH 7.4, 30°C, mutant A106T/Q189K/T399S/R409L, mutant D153V/I214F
0.251
progesterone
pH 7.4, 30°C, for determination of Km-value the formation of the product 15beta-hydroxy-progesterone is determined
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3.3
Ferredoxin
Bacillus megaterium
Q06069
pH 7.4, 30°C, wild-type ferredoxin from the cyanobacterium Anabaena PCC 7119
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1.34
flavodoxin
Bacillus megaterium
Q06069
pH 7.4, 30°C, wild-type flavodoxin from the cyanobacterium Anabaena PCC 7119
-
additional information
additional information
Bacillus megaterium
Q06069
turnover-number for the deoxicorticosterone 15beta-hydroxylase activity of recombinant BmCYP106A2 when using the NADPH/Anabaena ferredoxin electron donor system and different electron carrier proteins. Use of different AnFld mutants at positions W57, I59 and I92, which alter their aromatic and hydrophobic character, as electron donor to BmCYP106A2
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
the Vmax values for 15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone are determined as 337.3, 22.3, 17.5, and 6.5 nmol product/min/nmol CYP106A2
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.4
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
by adding the ligands imidazole and metyrapone, it is not possible to prevent CYP106A2 from degradation in the crystallization process
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
it is possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing flavoprotein (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system is fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin has an apparent sulfur to iron ratio of 0.98 and shows g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic resonance spectroscopy
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the purification procedure includes chromatography on DEAE-cellulose, Ultrogel ACA-54, DEAE-Sepharose, octyl-Sepharose, and hydroxyapatite
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a steroid 15beta-hydroxylating whole-cell solvent tolerant biocatalyst is constructed by expressing the Bacillus megaterium steroid hydroxylase CYP106A2 in the solvent tolerant Pseudomonas putida S12
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CYP106A2 can be easily expressed in Escherichia coli with a high yield and can be reconstituted using the adrenal redox proteins, adrenodoxin and adrenodoxin reductase
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expressed in Escherichia coli and Bacillus subtilis. No hydroxylation is found with protein extracts from recombinant Escherichia coli strains since cytochrome P450meg needs additional electron transfer proteins for enzymatic activity, which are missing in Escherichia coli. Bacillus subtilis, in contrast to Escherichia coli, contains an electron transfer system capable of supporting the activity of cytochrome P450meg
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expression in Escherichia coli, wild type and mutant S72A/V73I, the DELTA72 mutant, which lacks the first 72 amino acids, is not expressed in Escherichia coli at a detectable amount, suggesting that the truncated mutant cannot fold properly within the bacterial cell
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expression of the enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450-catalyzed hydroxylations are identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations a new concept for CYP106A2-catalyzed steroid hydroxylations is developed by which the productivity of progesterone and testosterone conversion could be increased up to 18fold to yield an absolute productivity up to 5.5 g/L*d
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functional expression of CYP106A2 in Escherichia coli strain BL21 from plasmid pET-CYP13 on the outer membrane with exposure on the surface without the external addition of the heme group but absolutely requiring the coexpression of TolC channel protein JW5503, because Escherichia coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme
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recombinant expression of CYP106A2 by protoplast transformation is only successfully in the plasmid-less Bacillus megaterium strain MS941, not in strain ATCC 13368, coexpression of heterologous redox chain of the P450, bovine adrenodoxin reductase, and bovine adrenodoxin
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A106T
-
kcat/Km for conversion of 11-deoxycortisol is 3.17fold higher than wild-type value, 1.43fold increase in progesterone conversion
A106T/Q189K/T399S/R409L
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kcat/Km for conversion of 11-deoxycortisol is 4.3fold higher than wild-type value, 1.13fold increase in progesterone conversion
A106T/R409L
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kcat/Km for conversion of 11-deoxycortisol is 3fold higher than wild-type value, 1.43fold increase in progesterone conversion
D153V/I214F
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kcat/Km for conversion of 11-deoxycortisol is 2.64fold higher than wild-type value, progesterone conversion is 93% of wild-type value
D217V
-
kcat/Km for conversion of 11-deoxycortisol is 2.68fold higher than wild-type value, 1.18fold increase in progesterone conversion
E90V/D185G
-
Vmax/Km for progesterone is 1.3fold higher than wild-type value
G397P
-
the mutant exhibits 2% of the wild-type activity, Vmax/Km for progesterone is 51.7fold lower than wild-type value
K27R/I71T/I215T
-
Vmax/Km for progesterone is 2.5fold higher than wild-type value
S72A/V73I
-
mutant does not show a better stability in the crystallization process than the wild-type protein
T396R
-
mutant does not produce any hydroxylated product up to an adrenodoxin concentration of 0.1 mM
additional information
additional information
-
modelling of CYP106A2 and site-directed mutagenesis of the protein to check the accuracy of the computer-derived model of CYP106A2
additional information
-
recombinant reconstitution of the whole cell conversion of 11-keto-beta-boswellic acid in Bacillus megaterium strain MS941 by coexpression of heterologous redox chain of the P450, bovine adrenodoxin reductase, and bovine adrenodoxin, as well as a a NADPH-regenerating system
additional information
-
construction of a CYP106A2 knockout strain; recombinant reconstitution of the whole cell conversion of 11-keto-beta-boswellic acid in Bacillus megaterium strain MS941 by coexpression of heterologous redox chain of the P450, bovine adrenodoxin reductase, and bovine adrenodoxin, as well as a a NADPH-regenerating system
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
synthesis
-
efficient approach towards the preparative scale synthesis of hydroxylated steroid derivatives. Improve CYP106A2-catalyzed steroid hydroxylation towards higher productivity and quantitative product formation. Because substrate transport into the cell limits the whole-cell biotransformation, activity can be increased sixfold by using membrane-free crude cell as biocatalyst
synthesis
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a steroid 15beta-hydroxylating whole-cell solvent tolerant biocatalyst is constructed by expressing the Bacillus megaterium steroid hydroxylase CYP106A2 in the solvent tolerant Pseudomonas putida S12. Testosterone hydroxylation is improved by a factor 16 by co-expressing Fer, a putative Fe-S protein from Bacillus subtilis. The specificity for 15beta-hydroxylation is improved by mutating threonine residue 248 of CYP106A2 into valine. These insights provide the basis for an optimized whole-cell steroid-hydroxylating biocatalyst that can be applied with an organic solvent phase
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LINKS TO OTHER DATABASES (specific for EC-Number 1.14.15.8)
Information
