Information on EC 1.14.13.54 - ketosteroid monooxygenase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.14.13.54
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RECOMMENDED NAME
GeneOntology No.
ketosteroid monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
17alpha-hydroxyprogesterone + NADPH + H+ + O2 = androstenedione + acetate + NADP+ + H2O
show the reaction diagram
(3)
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a ketosteroid + NADPH + H+ + O2 = a steroid ester/lactone + NADP+ + H2O
show the reaction diagram
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androstenedione + NADPH + H+ + O2 = testololactone + NADP+ + H2O
show the reaction diagram
(2)
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progesterone + NADPH + H+ + O2 = testosterone acetate + NADP+ + H2O
show the reaction diagram
(1)
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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reduction
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SYSTEMATIC NAME
IUBMB Comments
ketosteroid,NADPH:oxygen oxidoreductase (20-hydroxylating, ester-producing/20-hydroxylating, side-chain cleaving/17-hydroxylating, lactonizing)
A single FAD-containing enzyme catalyses three types of monooxygenase (Baeyer-Villiger oxidation) reaction. The oxidative esterification of a number of derivatives of progesterone to produce the corresponding 17alpha-hydroxysteroid 17-acetate ester, such as testosterone acetate, is shown in Reaction (1). The oxidative lactonization of a number of derivatives of androstenedione to produce the 13,17-secoandrosteno-17,13alpha-lactone, such as testololactone, is shown in Reaction (2). The oxidative cleavage of the 17beta-side-chain of 17alpha-hydroxyprogesterone to produce androstenedione and acetate is shown in Reaction (3). Reaction (1) is also catalysed by EC 1.14.99.4 (progesterone monooxygenase), and Reactions (2) and (3) correspond to that catalysed by EC 1.14.99.12 (androst-4-ene-3,17-dione monooxygenase). The possibility that a single enzyme is responsible for the reactions ascribed to EC 1.14.99.4 and EC 1.14.99.12 in other tissues cannot be excluded.
CAS REGISTRY NUMBER
COMMENTARY hide
9044-53-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain ATCC 13368
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Manually annotated by BRENDA team
ATTC 11011
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,4-Pregnadiene-17alpha,21-diol-3,11,20-trione + NADPH + O2
?
show the reaction diagram
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-
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11-deoxycortisol + NADPH + O2
? + NADP+ + H2O
show the reaction diagram
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?
11beta-Hydroxyprogesterone + NADPH + O2
?
show the reaction diagram
17alpha-hydroxypregnenolone + NADPH + O2
?
show the reaction diagram
17alpha-Hydroxyprogesterone + NADPH + O2
Androstenedione + acetate + NADP+ + H2O
show the reaction diagram
18-Hydroxydeoxycorticosterone + NADPH + O2
?
show the reaction diagram
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-
-
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21-Deoxycortisol + NADPH + O2
?
show the reaction diagram
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Aldosterone + NADPH + O2
?
show the reaction diagram
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androstenedione + NADPH + O2
3-oxo-13,17-secoandrost-4-eno-17,13-alpha-lactone + NADP+ + H2O
show the reaction diagram
Corticosterone + NADPH + O2
?
show the reaction diagram
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Cortisol + NADPH + O2
?
show the reaction diagram
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Cortisone + NADPH + O2
?
show the reaction diagram
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deoxycorticosterone + NADPH + O2
?
show the reaction diagram
phenylacetone + NADPH + H+ + O2
benzylacetate + NADP+ + H2O
show the reaction diagram
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?
Pregnenolone + NADPH + O2
?
show the reaction diagram
progesterone + NADPH + H+ + O2
testosterone acetate + NADP+ + H2O
show the reaction diagram
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?
Progesterone + NADPH + O2
Testosterone acetate + NADP+ + H2O
show the reaction diagram
additional information
?
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the enzyme does not accept 3beta-hydroxy-5-ene steroids as substrates
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
progesterone + NADPH + H+ + O2
testosterone acetate + NADP+ + H2O
show the reaction diagram
O50641
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?
Progesterone + NADPH + O2
Testosterone acetate + NADP+ + H2O
show the reaction diagram
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5-Pregnene-3beta,20alpha-diol
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lactonization of androstenedione
pregnenolone
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and other C21-20-ketosteroids which are the substrates for oxygenative esterification of the enzyme, strongly inhibit lactonization of androstenedione
progesterone
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strong, competitive inhibitor of lactonization of 17-ketosteroids
testosterone
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lactonization of androstenedione
Testosterone acetate
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lactonization of androstenedione
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04
androstenedione
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0.00044 - 0.0043
NADPH
0.07
O2
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with progesterone and NADPH as cosubstrates
0.2 - 2.6
phenylacetone
0.0004 - 0.055
progesterone
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2 - 3
phenylacetone
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.2 - 2.6
phenylacetone
3713
0.1 - 5.2
progesterone
286
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.95
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purified enzyme
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
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androstenedione monooxygenase reaction
7.8
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progesterone monooxygenase reaction
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8
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pH 5.5: about 20% of maximal activity, pH 8.0: about 40% of maximal activity, androstenedione monooxygenase activity
6 - 9
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pH 6.0: about 35% of maximal activity, pH 9.0: about 65% of maximal activity, progesterone monooxygenase activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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progesterone monooxygenase activity
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
116000
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gel filtration, gel electrophoresis
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 60000, SDS-PAGE; x * 60133, calculated from the deduced amino acid sequence; x * 60919, holoenzyme, calculated from the deduced amino acid sequence
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, using 1.8-2.2 M MgSO4, 0.1 M MES/HCl, pH 6.2-6.5
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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half-life: 8 days
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, Tris buffer, stable for more than 6 months, enzyme in crude extract
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni2+-affinity column chromatography and Superdex 75 gel filtration
recombinant enzyme from Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3)
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expressed in Escherichia coli BL21(DE3) CodonPlus cells
expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K295A
the mutant is 3times more active than the wild type enzyme
L500Y
the mutant is about 2.5times more active than the wild type enzyme
P157Q
the mutant shows wild type activity
T345L
the mutation turns the enzyme inactive against progesterone without altering the catalytic efficiency for phenylacetone
V291A
the mutant is less active than the wild type enzyme
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reconstitution of recombinant protein using adrenodoxin and adrenodoxin reductase
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