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Information on EC 1.14.15.29 - cholest-4-en-3-one 26-monooxygenase [(25S)-3-oxocholest-4-en-26-oate forming] and Organism(s) Mycobacterium tuberculosis and UniProt Accession P9WPP0

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IUBMB Comments
A cytochrome P-450 (heme-thiolate) protein found in several bacterial pathogens. The enzyme is involved in degradation of the host's 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 (25S) configuration. The enzyme is part of a two-component system that also includes a ferredoxin reductase (most likely KshB, which also interacts with EC 1.14.15.30, 3-ketosteroid 9alpha-monooxygenase). The enzyme also accepts cholesterol as a substrate. cf. EC 1.14.15.28, cholest-4-en-3-one 27-monooxygenase.
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This record set is specific for:
Mycobacterium tuberculosis
UNIPROT: P9WPP0
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The taxonomic range for the selected organisms is: Mycobacterium tuberculosis
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
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cholest-4-en-3-one
+
3
NADH
+
3
H+
+
3
O2
=
(25S)-3-oxocholest-4-en-26-oate
+
3
NAD+
+
4
H2O
+
3
+
3
+
3
=
+
3
+
4
Synonyms
cyp125, cyp125a1, cyp125a3, steroid c26-monooxygenase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cholest-4-en-3-one 26-monooxygenase
-
-
-
-
CYP125A1
cytochrome P450 125
-
-
steroid C26-monooxygenase
-
-
SYSTEMATIC NAME
IUBMB Comments
cholest-4-en-3-one,[reduced ferredoxin]:oxygen oxidoreductase [(25S)-3-oxocholest-4-en-26-oate forming]
A cytochrome P-450 (heme-thiolate) protein found in several bacterial pathogens. The enzyme is involved in degradation of the host's 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 [4]. The products are exclusively in the (25S) configuration. The enzyme is part of a two-component system that also includes a ferredoxin reductase (most likely KshB, which also interacts with EC 1.14.15.30, 3-ketosteroid 9alpha-monooxygenase). The enzyme also accepts cholesterol as a substrate. cf. EC 1.14.15.28, cholest-4-en-3-one 27-monooxygenase.
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
27-hydroxycholest-4-en-3-one + 4 reduced ferredoxin [iron-sulfur] cluster + 4 H+ + O2
cholest-4-en-3-one-27-oic acid + 4 oxidized ferredoxin [iron-sulfur] cluster + 3 H2O
show the reaction diagram
-
-
-
?
cholest-4-en-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
27-hydroxycholest-4-en-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
physiological substrate, the enzyme hydroxylates cholest-4-en-3-one at C-27
-
-
?
(25S)-25-bromo-27-norcholesterol + 6 reduced [2Fe-2S] ferredoxin + 3 O2
(25S)-25-bromo-27-norcholesterol-26-oate + 6 oxidized [2Fe-2S] ferredoxin + 4 H2O
show the reaction diagram
-
-
-
?
26-methylcholesterol + 6 reduced [2Fe-2S] ferredoxin + 3 O2
(25S)-3beta-hydroxycholest-5-en-27-oate + 6 oxidized [2Fe-2S] ferredoxin + 4 H2O
show the reaction diagram
-
-
-
?
cholest-4-en-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
(25S)-26-hydroxycholest-4-en-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
-
-
CYP125A1 generates oxidized sterols of the (25S)-26-hydroxy configuration
-
?
cholest-4-en-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
26-hydroxycholest-4-en-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
cholest-5-en-3-beta-ol + 6 reduced [2Fe-2S] ferredoxin + 3 O2
(25S)-3beta-hydroxycholest-5-en-26-oate + 6 oxidized [2Fe-2S] ferredoxin + 4 H2O
show the reaction diagram
-
-
-
?
cholesterol + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
27-hydroxycholesterol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
-
-
-
-
?
cholesterol + reduced ferredoxin [iron-sulfur] cluster + O2
26-hydroxycholesterol + oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
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
27-hydroxycholest-4-en-3-one + 4 reduced ferredoxin [iron-sulfur] cluster + 4 H+ + O2
cholest-4-en-3-one-27-oic acid + 4 oxidized ferredoxin [iron-sulfur] cluster + 3 H2O
show the reaction diagram
-
-
-
?
cholest-4-en-3-one + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
27-hydroxycholest-4-en-3-one + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
physiological substrate, the enzyme hydroxylates cholest-4-en-3-one at C-27
-
-
?
cholest-5-en-3-beta-ol + 6 reduced [2Fe-2S] ferredoxin + 3 O2
(25S)-3beta-hydroxycholest-5-en-26-oate + 6 oxidized [2Fe-2S] ferredoxin + 4 H2O
show the reaction diagram
-
-
-
?
additional information
?
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
alpha-[(4-methylcyclohexyl)carbonyl amino]-N-4-pyridinyl-1H-indole-3-propanamide
LP10, 30% inhibition at 0.2 mM
nitric oxide
-
reversible inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0208
cholest-4-en-3-one
-
pH and temperature not specified in the publication
0.0107
cholesterol
-
pH and temperature not specified in the publication
1.2
O2
-
apparent value, Km above 1.2 mM, in 0.1 M potassium phosphate at pH 7.0, temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.3
(25S)-25-bromo-27-norcholesterol
pH 7.5, 25°C
0.317
26-methylcholesterol
pH 7.5, 25°C
2.92
cholest-4-en-3-one
-
pH and temperature not specified in the publication
0.467
cholest-5-en-3-beta-ol
pH 7.5, 25°C
0.47
cholesterol
-
pH and temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11000
O2
-
apparent value, in 0.1 M potassium phosphate at pH 7.0, temperature not specified in the publication
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
CYP125A1 is a key enzyme in cholesterol metabolism and plays a crucial role in circumventing the deleterious effect of cholest-4-en-3-one
physiological function
CYP125A1 is required to incorporate the cholesterol side chain carbon atoms into cellular lipids. CYP125A1 is essential for growth of CDC1551 in media containing cholesterol or cholest-4-en-3-one
physiological function
-
Cyp125 is not essential for the growth of Mycobacterium tuberculosis on cholesterol
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46400
-
MALDI-TOF mass spectrometry
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme bound to cholest-4-en-3-one, hanging drop vapor diffusion method, using 0.2 M ammonium sulfate, 0.1 M bis-Tris, pH 5.5, and 25% (w/v) PEG 3350
hanging drop vapor diffusion method, using 2.0 M ammonium sulfate, 0.1 M bis-Tris (pH 5.5) and 0.5 M NaCl (truncated enzyme C429L in complex with inhibitor) or 0.1 M ammonium acetate, 0.1 M bis-Tris (pH 5.5) and 17% PEG 10000, or 2.0 M ammonium sulfate and 0.1 M Tris HCl pH 8.5 (truncated enzyme C429L without any ligand)
sitting drop vapor diffusion method, using MgCl2 with 0.1 M HEPES, pH 7.0 or 7.5, and 20% (w/v) PEG 6000 or 25% (w/v) PEG 3350, respectively
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C429L
the modification affect neither the binding nor the catalytic properties of the enzyme
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
-
the enzyme is heat inactivated by incubation at 65°C for 30 min
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-NTA resin column chromatography, Resource-Q column chromatography, and Sephacryl S-200 gel filtration
-
Ni2+-affinity column chromatography and Q Sepharose column chromatography
-
Source 15Q column chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli DH5alpha cells
expressed in Escherichia coli
-
expressed in Escherichia coli DH5alpha cells
-
expressed in Escherichia coli HMS174 (DE3) cells
-
expressed in Rhodococcus jostii strain RHA1
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ouellet, H.; Lang, J.; Couture, M.; De Montellano, P.
Reaction of Mycobacterium tuberculosis Cytochrome P450 enzymes with nitric oxide
Biochemistry
48
863-872
2009
Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv
Manually annotated by BRENDA team
Ouellet, H.; Kells, P.; Ortiz De Montellano, P.; Podust, L.
Reverse type I inhibitor of Mycobacterium tuberculosis CYP125A1
Bioorg. Med. Chem. Lett.
21
332-337
2011
Mycobacterium tuberculosis (P9WPP1), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WPP1)
Manually annotated by BRENDA team
McLean, K.; Lafite, P.; Levy, C.; Cheesman, M.; Mast, N.; Pikuleva, I.; Leys, D.; Munro, A.
The structure of Mycobacterium tuberculosis CYP125: Molecular basis for cholesterol binding in a P450 needed for host infection
J. Biol. Chem.
284
35524-35533
2009
Mycobacterium tuberculosis
Manually annotated by BRENDA team
Capyk, J.; Kalscheuer, R.; Stewart, G.; Liu, J.; Kwon, H.; Zhao, R.; Okamoto, S.; Jacobs Jr., W.; Eltis, L.; Mohn, W.
Mycobacterial cytochrome P450 125 (Cyp125) catalyzes the terminal hydroxylation of C27 steroids
J. Biol. Chem.
284
35534-35542
2009
Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis variant bovis, Mycobacterium tuberculosis variant bovis bacillus Calmette-Guerin
Manually annotated by BRENDA team
Johnston, J.B.; Ouellet, H.; Ortiz de Montellano, P.R.
Functional redundancy of steroid C26-monooxygenase activity in Mycobacterium tuberculosis revealed by biochemical and genetic analyses
J. Biol. Chem.
285
36352-36360
2010
Mycobacterium tuberculosis, Mycobacterium tuberculosis CDC 1551, Mycobacterium tuberculosis H37Rv
Manually annotated by BRENDA team
Ouellet, H.; Guan, S.; Johnston, J.; Chow, E.; Kells, P.; Burlingame, A.; Cox, J.; Podust, L.; De Montellano, P.
Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one
Mol. Microbiol.
77
730-742
2010
Mycobacterium tuberculosis (P9WPP0), Mycobacterium tuberculosis, Mycobacterium tuberculosis CDC 1551 (P9WPP0)
Manually annotated by BRENDA team
Johnston, J.B.; Singh, A.A.; Clary, A.A.; Chen, C.K.; Hayes, P.Y.; Chow, S.; De Voss, J.J.; Ortiz de Montellano, P.R.
Substrate analog studies of the omega-regiospecificity of Mycobacterium tuberculosis cholesterol metabolizing cytochrome P450 enzymes CYP124A1, CYP125A1 and CYP142A1
Bioorg. Med. Chem.
20
4064-4081
2012
Mycobacterium tuberculosis (P9WPP1), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WPP1)
Manually annotated by BRENDA team