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Information on EC 1.14.15.8 - steroid 15beta-monooxygenase and Organism(s) Priestia megaterium and UniProt Accession Q06069

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IUBMB Comments
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 .
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This record set is specific for:
Priestia megaterium
UNIPROT: Q06069
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Word Map
The taxonomic range for the selected organisms is: Priestia megaterium
The expected taxonomic range for this enzyme is: Priestia megaterium
Synonyms
cyp106a1, 15beta-hydroxylase, cytochrome p-450meg, cytochrome p450meg, bmcyp106a2, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15beta-hydroxylase
-
15beta-hydroxylase CYP106A2
-
cytochrome P-450meg
-
cytochrome P450meg
-
steroid-15-beta-monooxygenase
-
cytochrome P-450meg
-
-
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].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
11-deoxycorticosterone + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + 7beta,15beta-dihydroxy-11-deoxycorticosterone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
11-deoxycorticosterone + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized adrenodoxin + H2O
show the reaction diagram
11-deoxycorticosterone + reduced ferredoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized ferredoxin + H2O
show the reaction diagram
ferredoxin from the cyanobacterium Anabaena PCC 7119
-
-
?
11-deoxycorticosterone + reduced flavodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized flavodoxin + H2O
show the reaction diagram
flavodoxin from the cyanobacterium Anabaena PCC 7119
-
-
?
11-deoxycortisol + reduced adrenodoxin + O2
15beta-15,17,21-trihydroxypregn-4-ene-3,20-dione + oxidized adrenodoxin + H2O
show the reaction diagram
adrenodoxin is a [2Fe-2S] ferredoxin involved in electron transfer from NADPH+ (via a reductase) to cytochrome P-450 in the adrenal gland
-
-
?
11-deoxycortisol + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycortisol + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
11beta-hydroxy-4-androstene-3,17-dione + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
17alpha-methyltestosterone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
19-nortestosterone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
4-androstene-3,17-dione + reduced adrenodoxin + O2
15beta-hydroxyandrostene-3,17-dione + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
6alpha-fluoro-16alpha-methyl-deoxycorticosterone + reduced ferredoxin + O2
15beta-hydroxy-6alpha-fluoro-16alpha-methyl-deoxycorticosterone + oxidized ferredoxin + H2O
show the reaction diagram
-
-
-
?
abietic acid + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
binding of abietic acid results in a type II difference spectrum typical for nitrogenous inhibitors
-
-
?
corticosterone + reduced adrenodoxin + O2
15beta-hydroxycorticosterone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
cortisone + reduced adrenodoxin + O2
15beta-hydroxycortisone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
dehydroabietic acid + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
dehydroepiandrosterone + reduced adrenodoxin + O2
7beta-hydroxy-dehydroepiandrosterone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
deoxycorticosterone + reduced ferredoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized ferredoxin + H2O
show the reaction diagram
-
-
-
r
deoxycorticosterone + reduced flavodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized flavodoxin + H2O
show the reaction diagram
-
-
-
r
dexamethasone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
digitoxigenin + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
isopimaric acid + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
prednisolone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
prednisone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
pregnenolone + reduced adrenodoxin + O2
7beta-hydroxy-pregnenolone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
progesterone + reduced adrenodoxin + O2
15beta-hydroxy-progesterone + 11alpha-hydroxy-progesterone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
progesterone + reduced adrenodoxin + O2
15beta-hydroxy-progesterone + oxidized adrenodoxin + H2O
show the reaction diagram
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
-
-
?
progesterone + reduced adrenodoxin + O2
15beta-hydroxyprogesterone + oxidized adrenodoxin + H2O
show the reaction diagram
progesterone + reduced ferredoxin + O2
15beta-hydroxy-progesterone + oxidized ferredoxin + H2O
show the reaction diagram
Bacillus megaterium ferredoxin may be replaced by adrenal ferredoxin
-
-
?
progesterone + reduced megaredoxin + O2
15beta-hydroxyprogesterone + oxidized megaredoxin + H2O
show the reaction diagram
-
-
-
?
testosterone + reduced adrenodoxin + O2
15beta-hydroxytestosterone + oxidized adrenodoxin + H2O
show the reaction diagram
11-deoxycorticosterone + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + 7beta,15beta-dihydroxy-11-deoxycorticosterone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
11-deoxycorticosterone + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
-
?
11-deoxycorticosterone + reduced megaredoxin + O2
15beta-hydroxy-11-deoxycorticosterone + oxidized megaredoxin + H2O
show the reaction diagram
-
megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
-
-
?
11-deoxycortisol + reduced adrenodoxin + O2
15beta-hydroxy-11-deoxycortisol + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
11-oxo-beta-boswellic acid + reduced ferredoxin + O2
(3alpha,15beta)-3,15-dihydroxy-11-oxours-12-en-24-oic acid + H2O
show the reaction diagram
-
a pentacyclic triterpene, 15beta-hydroxylation
-
-
?
11beta-hydroxy-4-androstene-3,17-dione + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
17alpha-hydroxyprogesterone + reduced megaredoxin + O2
(15beta,17alpha)-15,17-dihydroxyprogesterone + oxidized megaredoxin + H2O
show the reaction diagram
-
megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
-
-
?
17alpha-methyltestosterone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
19-nortestosterone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
20alpha-dihydroprogesterone + reduced megaredoxin + O2
(15beta,20alpha)-15-hydroxy-20-dihydroprogesterone + oxidized megaredoxin + H2O
show the reaction diagram
-
megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
-
-
?
4-androstene-3,17-dione + reduced megaredoxin + O2
15beta-hydroxyandrostene-3,17-dione + oxidized megaredoxin + H2O
show the reaction diagram
-
megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
-
-
?
abietic acid + reduced adrenodoxin + O2
12-hydroxyabietic acid + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
-
?
abietic acid + reduced ferredoxin + O2
?
show the reaction diagram
-
a pentacyclic triterpene
-
-
?
corticosterone + reduced megaredoxin + O2
15beta-hydroxycorticosterone + oxidized megaredoxin + H2O
show the reaction diagram
-
megaredoxin is an iron-sulfur protein of Bacillus megaterium, preferred substrate, megaredoxin has an apparent sulfur to iron ratio of 0.98
-
-
?
cortisone + reduced adrenodoxin + O2
15beta-hydroxycortisone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
dexamethasone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
imipramine + reduced adrenodoxin + O2
desipramine + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
-
?
prednisolone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
prednisone + reduced adrenodoxin + O2
? + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
progesterone + reduced megaredoxin + O2
15beta-hydroxyprogesterone + oxidized megaredoxin + H2O
show the reaction diagram
-
megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
-
-
?
testosterone + reduced acceptor + O2
15beta-hydroxytestosterone + oxidized acceptor + H2O
show the reaction diagram
-
the major product is identified as 15beta-hydroxytestosterone. 6beta-Hydroxytestosterone and androst-4-ene-3,17-dione are present as minor products
-
-
?
testosterone + reduced adrenodoxin + O2
15beta-hydroxytestosterone + oxidized adrenodoxin + H2O
show the reaction diagram
-
-
-
?
testosterone + reduced megaredoxin + O2
15beta-hydroxytestosterone + oxidized megaredoxin + H2O
show the reaction diagram
-
megaredoxin is an iron-sulfur protein of Bacillus megaterium, megaredoxin has an apparent sulfur to iron ratio of 0.98
-
-
?
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
additional information
?
-
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
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
adrenodoxin
-
dependent on
-
cytochrome P450
-
-
-
cytochrome P450meg
-
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
-
Ferredoxin
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Iron
deoxycorticosterone binds in the heme pocket near the iron ligand
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11beta-hydroxyprogesterone
-
-
16alpha-hydroxyprogesterone
-
-
17alpha,21-dihydroxyprogesterone
-
-
17alpha-hydroxyprogesterone
-
-
19-norandrostenedione
-
-
19-nortestosterone
-
-
20alpha-dihydroprogesterone
-
-
deoxycorticosterone
-
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
phosphatidylcholine
stimulates
heme
-
the heme content of cytochrome P-450meg is 0.94 nmol of heme per nmol of cytochrome P-450
Iron
-
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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.037 - 0.093
11-deoxycortisol
0.0012 - 0.0056
deoxycorticosterone
0.0007 - 0.251
progesterone
0.00314
reduced ferredoxin
pH 7.4, 30°C, wild-type ferredoxin from the cyanobacterium Anabaena PCC 7119
0.00734
reduced flavodoxin
pH 7.4, 30°C, wild-type flavodoxin from the cyanobacterium Anabaena PCC 7119
0.56
17alpha-hydroxyprogesterone
-
22°C
0.45
20alpha-dihydroprogesterone
-
22°C
0.625
4-androstene-3,17-dione
-
22°C
0.095
corticosterone
-
22°C
0.06
deoxycorticosterone
-
22°C
0.143
progesterone
-
22°C
1.11
testosterone
-
22°C
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.3
reduced ferredoxin
pH 7.4, 30°C, wild-type ferredoxin from the cyanobacterium Anabaena PCC 7119
1.34
reduced flavodoxin
pH 7.4, 30°C, wild-type flavodoxin from the cyanobacterium Anabaena PCC 7119
additional information
additional information
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.07
11beta-hydroxyprogesterone
-
22°C
0.05
16alpha-hydroxyprogesterone
-
22°C
0.14
17alpha,21-dihydroxyprogesterone
-
22°C
0.055
17alpha-hydroxyprogesterone
-
22°C
0.07
19-norandrostenedione
-
22°C
0.16
19-nortestosterone
-
22°C
0.05
20alpha-dihydroprogesterone
-
22°C
0.11
deoxycorticosterone
-
22°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
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
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
assay at
30
-
assay at
37
-
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.9
isoelectric focusing
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
CPXM_PRIMG
410
0
46956
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
52000
x * 52000, SDS-PAGE
40000
-
sucrose density gradient centrifugation
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
substrate-free enzyme to 1.8 A resolution, cocrystallization of CYP106A2 with abietic acid reveals bending of the heme cofactor when abietic acid is bound in the active site
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
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
kcat/Km for conversion of 11-deoxycortisol is 4.3fold higher than wild-type value, 1.13fold increase in progesterone conversion
A106T/R409L
kcat/Km for conversion of 11-deoxycortisol is 3fold higher than wild-type value, 1.43fold increase in progesterone conversion
A243S
mutant engineered for steroid 9alpha- and 6beta-hydroxylation, 10fold increase in the selectivity towards 6alpha-hydroxylation of progesterone. Mutant produces 3.4% 15beta-hydroxy-progesterone plus 4.6% 11alpha-hydroxy-progesterone plus 2.4% 9alpha-hydroxy-progesterone plus 82.7% 6beta-hydroxy-progesterone
A395L
Vmax/Km for progesterone is 2fold lower than wild-type value
D153V/I214F
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
F165I
1.25fold increase in progesterone conversion
F165L/A395E/G397V
mutant engineered for steroid 9alpha- and 6beta-hydroxylation, 10fold increase in the selectivity towards progesterone 9lpha-hydroxylation. Mutant produces 11.3% 15beta-hydroxy-progesterone plus 17.2% 11alpha-hydroxy-progesterone plus 59.7% 9alpha-hydroxy-progesterone plus 6.8% 6beta-hydroxy-progesterone
G397P
the mutant exhibits 2% of the wild-type activity, Vmax/Km for progesterone is 51.7fold lower than wild-type value
I86T
Vmax/Km for progesterone is 2fold higher than wild-type value
K27R/I71T/I215T
Vmax/Km for progesterone is 2.5fold higher than wild-type value
Q398S
Vmax/Km for progesterone is 5fold lower than wild-type value
S394I
Vmax/Km for progesterone is 7.5fold lower 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
T248V
-
specificity for 15beta-hydroxylation is improved
additional information
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
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, 4 weeks, degradation during storage
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
the purification procedure includes chromatography on DEAE-cellulose, Ultrogel ACA-54, DEAE-Sepharose, octyl-Sepharose, and hydroxyapatite
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
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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
expression in Escherichia coli
expression in Escherichia coli BL21DE3
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
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
exression in Bacillus megaterium
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
-
expression in Escherichia coli
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
-
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
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
synthesis
-
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Virus, C.; Bernhardt, R.
Molecular evolution of a steroid hydroxylating cytochrome P450 using a versatile steroid detection system for screening
Lipids
43
1133-1141
2008
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Simgen, B.; Contzen, J.; Schwarzer, R.; Bernhardt, R.; Jung, C.
Substrate binding to 15beta-hydroxylase (CYP106A2) probed by FT infrared spectroscopic studies of the iron ligand CO stretch vibration
Biochem. Biophys. Res. Commun.
269
737-742
2000
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Lisurek, M.; Kang, M.J.; Hartmann, R.W.; Bernhardt, R.
Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry
Biochem. Biophys. Res. Commun.
319
677-682
2004
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Virus, C.; Lisurek, M.; Simgen, B.; Hannemann, F.; Bernhardt, R.
Function and engineering of the 15beta-hydroxylase CYP106A2
Biochem. Soc. Trans.
34
1215-1218
2006
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Goni, G.; Zllner, A.; Lisurek, M.; Velazquez-Campoy, A.; Pinto, S.; Gomez-Moreno, C.; Hannemann, F.; Bernhardt, R.; Medina, M.
Cyanobacterial electron carrier proteins as electron donors to CYP106A2 from Bacillus megaterium ATCC 13368
Biochim. Biophys. Acta
1794
1635-1642
2009
Priestia megaterium (Q06069), Priestia megaterium
Manually annotated by BRENDA team
Zehentgruber, D.; Hannemann, F.; Bleif, S.; Bernhardt, R.; Ltz, S.
Towards preparative scale steroid hydroxylation with cytochrome P450 monooxygenase CYP106A2.
Chembiochem
11
713-721
2010
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Lisurek, M.; Simgen, B.; Antes, I.; Bernhardt, R.
Theoretical and experimental evaluation of a CYP106A2 low homology model and production of mutants with changed activity and selectivity of hydroxylation
Chembiochem
16
1439-1449
2008
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Berg, A.; Gustafsson, J.A.; Ingelman-Sundberg, M.
Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium
J. Biol. Chem.
251
2831-2838
1976
Priestia megaterium
Manually annotated by BRENDA team
Berg, A.; Ingelman-Sundberg, M.; Gustafsson, M.
Purification and characterization of cytochrome P-450meg
J. Biol. Chem.
254
5264-5271
1979
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Ruijssenaars, H.J.; Sperling, E.M.; Wiegerinck, P.H.; Brands, F.T.; Wery, J.; de Bont, J.A.
Testosterone 15beta-hydroxylation by solvent tolerant Pseudomonas putida S12
J. Biotechnol.
131
205-208
2007
Priestia megaterium
Manually annotated by BRENDA team
Rauschenbach, R.; Isernhagen, M.; Noeske-Jungblut, C.; Boidol, W.; Siewert, G.
Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15 beta-monooxygenase from Bacillus megaterium ATCC 13368
Mol. Gen. Genet.
241
170-176
1993
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Bleif, S.; Hannemann, F.; Lisurek, M.; Von Kries, J.; Zapp, J.; Dietzen, M.; Antes, I.; Bernhardt, R.
Identification of CYP106A2 as a regioselective allylic bacterial diterpene hydroxylase
ChemBioChem
12
576-582
2011
Priestia megaterium (Q06069), Priestia megaterium ATCC 13368 (Q06069)
Manually annotated by BRENDA team
Bleif, S.; Hannemann, F.; Zapp, J.; Hartmann, D.; Jauch, J.; Bernhardt, R.
A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-beta-boswellic acid (KBA) based on a recombinant cytochrome P450 system
Appl. Microbiol. Biotechnol.
93
1135-1146
2012
Priestia megaterium, Priestia megaterium ATCC 13368
Manually annotated by BRENDA team
Schumacher, S.D.; Hannemann, F.; Teese, M.G.; Bernhardt, R.; Jose, J.
Autodisplay of functional CYP106A2 in Escherichia coli
J. Biotechnol.
161
104-112
2012
Priestia megaterium
Manually annotated by BRENDA team
Kiss, F.M.; Schmitz, D.; Zapp, J.; Dier, T.K.; Volmer, D.A.; Bernhardt, R.
Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium - identification of a novel 11-oxidase activity
Appl. Microbiol. Biotechnol.
99
8495-8514
2015
Priestia megaterium, Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Janocha, S.; Carius, Y.; Hutter, M.; Lancaster, C.R.; Bernhardt, R.
Crystal structure of CYP106A2 in substrate-free and substrate-bound form
ChemBioChem
17
852-860
2016
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Schmitz, D.; Zapp, J.; Bernhardt, R.
Steroid conversion with CYP106A2 - production of pharmaceutically interesting DHEA metabolites
Microb. Cell Fact.
13
81
2014
Priestia megaterium (Q06069)
Manually annotated by BRENDA team
Nikolaus, J.; Nguyen, K.T.; Virus, C.; Riehm, J.L.; Hutter, M.; Bernhardt, R.
Engineering of CYP106A2 for steroid 9alpha- and 6beta-hydroxylation
Steroids
120
41-48
2017
Priestia megaterium (Q06069)
Manually annotated by BRENDA team