Information on EC 1.14.13.221 - cholest-4-en-3-one 26-monooxygenase [(25R)-3-oxocholest-4-en-26-oate forming]

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The expected taxonomic range for this enzyme is: Mycobacterium

EC NUMBER
COMMENTARY hide
1.14.13.221
-
RECOMMENDED NAME
GeneOntology No.
cholest-4-en-3-one 26-monooxygenase [(25R)-3-oxocholest-4-en-26-oate forming]
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(25R)-26-hydroxycholest-4-en-3-one + NADPH + H+ + O2 = (25R)-26-oxocholest-4-en-3-one + NADP+ + 2 H2O
show the reaction diagram
(25R)-26-oxocholest-4-en-3-one + NADPH + H+ + O2 = (25R)-3-oxocholest-4-en-26-oate + NADP+ + H2O
show the reaction diagram
cholest-4-en-3-one + 3 NADPH + 3 H+ + 3 O2 = (25R)-3-oxocholest-4-en-26-oate + 3 NADP+ + 4 H2O
show the reaction diagram
cholest-4-en-3-one + NADPH + H+ + O2 = (25R)-26-hydroxycholest-4-en-3-one + NADP+ + H2O
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Microbial metabolism in diverse environments
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Steroid degradation
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SYSTEMATIC NAME
IUBMB Comments
cholest-4-en-3-one,NADPH:oxygen oxidoreductase [(25R)-3-oxocholest-4-en-26-oate forming]
This enzyme, found in several bacterial pathogens, is involved in degradation of the host cholesterol. It catalyses the hydroxylation of the C-26 carbon, followed by oxidation of the alcohol to the carboxylic acid via the aldehyde intermediate, initiating the degradation of the alkyl side-chain of cholesterol. The products are exclusively in the (25R) conformation. The enzyme also accepts cholesterol as a substrate. cf. EC 1.14.13.141, cholest-4-en-3-one 26-monooxygenase. The enzyme can receive electrons from ferredoxin reductase in vitro, its natural electron donor is not known yet.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(25R)-26-hydroxycholest-4-en-3-one + NADPH + H+ + O2
(25R)-26-oxocholest-4-en-3-one + NADP+ + 2 H2O
show the reaction diagram
-
-
-
-
?
(25R)-26-oxocholest-4-en-3-one + NADPH + H+ + O2
(25R)-3-oxocholest-4-en-26-oate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
cholest-4-en-3-one + 3 NADPH + 3 H+ + 3 O2
(25R)-3-oxocholest-4-en-26-oate + 3 NADP+ + 4 H2O
show the reaction diagram
cholest-4-en-3-one + 3 NADPH + 3 H+ + 3 O2 the enzyme
(25R)-3-oxocholest-4-en-26-oate + 3 NADP+ + 4 H2O
show the reaction diagram
cholest-4-en-3-one + NADPH + H+ + O2
(25R)-3-oxocholest-4-en-26-oate + NADP+ + H2O
show the reaction diagram
cholesterol + 3 NADPH + 3 H+ + 3 O2
26-carboxylic acid cholesterol
show the reaction diagram
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-
-
-
?
cholesterol + 3 NADPH + 3 H+ + 3 O2
?
show the reaction diagram
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
cholest-4-en-3-one + 3 NADPH + 3 H+ + 3 O2
(25R)-3-oxocholest-4-en-26-oate + 3 NADP+ + 4 H2O
show the reaction diagram
cholest-4-en-3-one + NADPH + H+ + O2
(25R)-3-oxocholest-4-en-26-oate + NADP+ + H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome P450
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cytochrome P450-dependent monooxygenase
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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the enzyme is relatively resistant to nitric oxide
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0103 - 0.0398
cholest-4-en-3-one
0.0077
cholesterol
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21°C, pH 7.4
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.4
cholest-4-en-3-one
Mycobacterium tuberculosis
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21°C, pH 7.4
0.278
cholesterol
Mycobacterium tuberculosis
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21°C, pH 7.4
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
118.6
cholest-4-en-3-one
Mycobacterium tuberculosis
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21°C, pH 7.4
5950
36.1
cholesterol
Mycobacterium tuberculosis
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21°C, pH 7.4
189
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with cholesteryl sulfate, sitting drop vapor diffusion method, using 20-25% PEG 3350, 10-13% (v/v) glycerol, and 50 mM MgCl2
sitting drop method, the CYP142 crystal structure is solved to 1.6 A
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
the substrate free enzyme is unstable and aggregates
736407
7
the P450 form of CYP142 is most stable at pH 7, and larger proportions of the P420 species are formed at the higher pH values, with near-complete P420 formation at pH 9
736407
8
the spectrum for the Fe2+-CO form is notably unstable, and the P450 species progressively collapses over time with P420 accumulation
736407
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
completely to the P450 state on binding of cholest-4-en-3-one at pH 8.0
stabilizing effect of substrate binding on the thiolate-coordinated CYP142, to the extent that the P420 form of CYP142 can be converted almost
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography, SP-Sepharose column chromatography, and Q-Sepharose column chromatography
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Ni2+-affinity column chromatography and Q Sepharose column chromatography
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nickel-nitrilotriacetic acid column chromatography and Q-Sepharose column chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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expressed in Escherichia coli DH5alpha cells