Information on EC 3.7.1.18 - 6-oxocamphor hydrolase

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The expected taxonomic range for this enzyme is: Rhodococcus

EC NUMBER
COMMENTARY hide
3.7.1.18
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RECOMMENDED NAME
GeneOntology No.
6-oxocamphor hydrolase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
bornane-2,6-dione + H2O = [(1S)-4-hydroxy-2,2,3-trimethylcyclopent-3-enyl]acetate
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
bornane-2,6-dione hydrolase
Isolated from Rhodococcus sp. The bornane ring system is cleaved by a retro-Claisen reaction to give the enol of alpha-campholonate. When separate from the enzyme the enol is tautomerised to the keto form as a 6:1 mixture of [(1S,3R)-2,2,3-trimethyl-4-oxocyclopentyl]acetate and [(1S,3S)-2,2,3-trimethyl-4-oxocyclopentyl]acetate.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene camK
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-allylbicyclo-[4.3.0]nonane-2,9-dione + H2O
?
show the reaction diagram
1-methylbicyclo-[4.3.0]nonane-2,9-dione + H2O
?
show the reaction diagram
1-methylbicyclo[5.3.0]decane-2,10-dione + H2O
?
show the reaction diagram
6-oxocamphor + H2O
(2R,4S)-alpha-campholinic acid
show the reaction diagram
6-oxocamphor + H2O
(2S,4S)-alpha-campholinic acid
show the reaction diagram
7,7-dimethylbicyclo[2.2.2]octane-2,6-dione + H2O
[(1S)-3,3-dimethyl-5-oxocyclohexyl]acetic acid
show the reaction diagram
8,8-dimethylbicyclo[2.2.2]octane-2,6-dione + H2O
[(1S)-2,2-dimethyl-5-oxocyclohexyl]acetic acid
show the reaction diagram
bicyclo[2.2.2]octan-2,6-dione + H2O
[(1S)-3-oxocyclohexyl]acetic acid
show the reaction diagram
bicyclo[2.2.2]octane-2,6-dione + H2O
?
show the reaction diagram
-
-
-
-
?
bornane-2,6-dione + H2O
[(1S)-4-hydroxy-2,2,3-trimethylcyclopent-3-enyl]acetate
show the reaction diagram
additional information
?
-
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
6-oxocamphor + H2O
(2S,4S)-alpha-campholinic acid
show the reaction diagram
bornane-2,6-dione + H2O
[(1S)-4-hydroxy-2,2,3-trimethylcyclopent-3-enyl]acetate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Zn2+ has a poor influence on enzyme activity, no effect by 1 M NaCl
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
72% inhibition at 1 mM
Hg2+
98% inhibition at 1 mM
Hydroxymercuribenzoate
14% inhibition at 1 mM
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N-ethylmaleimide
68% inhibition at 1 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
119% activity at 1 mM
additional information
the enzyme displays 25% higher activity in 50 mM phosphate buffer than 50 mM Tris/HCl at the same pH
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04 - 0.05
6-oxocamphor
additional information
additional information
-
kinetic analysis of OCH mutants, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
167 - 642.9
6-oxocamphor
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16100
6-oxocamphor
Rhodococcus sp.
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wild-type enzyme, pH and temperature not specified in the publication
15295
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
357.5
purified native enzyme, substrate 6-oxocamphor, pH 7.0, 25°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28488
3 * 28488, mass spectrometry
83000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
trimer
additional information
-
quaternary structure of OCH, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
selenomethionine-labeled enzyme, vapor diffusion hanging drop technique, protein solution containing 10 mg/ml protein in 50 mM Tris-HCl, pH 7.0, 1 mM dithiothreitol, 0.020 mM phenylmethylsulfonyl fluoride is mixed in a 1:1 ratio with reservoir solution, that contains for the native enzyme, the reservoir solution consisted of 0.1 M sodium acetate buffer, pH 4.5, 0.2 M ammonium sulfate, and 28% v/v PEG 4000, and for the selenomethionyl enzyme 0.1 M MES, pH 5.5, 0.2 M ammonium sulfate, 37.5% v/v 5,000 Da monomethyl ether, and 0.2% w/v n-octyl-beta-D-glucopyranoside, X-ray diffraction structure determination and analysis at 2.0-2.4 A resolution, selenomethionine multiple wave anomalous dispersion, molecular modeling
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OCH mutant H122A in complex with the minor diastereoisomer of (2S,4S)-alpha-campholinic acid, vapor diffusion hanging drop technique, mxing of protein solution containing 10 mg/ml protein in 50 mM Tris-HCl, pH 7.1, 1 mM dithiothreitol, and 0.02 mM phenylmethylsulfonyl fluoride with reservoir solution containing 0.1 M 2-(N-morpholino)ethanesulfonic acid, pH 5.6, 0.2 M calcium acetate, and 26% v/v PEG monomethyl ether 2000, in a 1:1 ratio, soakong of crystals in reservoir solution containing 6-oxocamphor for 30 min, X-ray diffraction structure determination and analysis at 1.9 A resolution, molecular replacement
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme 35.7fold from strain NCIMB 9784
recombinant enzyme from Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene camK, cloning and expression in Escherichia coli XL1 Blue
gene camK, expression in Escherichia coli strain BL21 (DE3)
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gene camK, expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) and B8434(DE3) and native and selenomethionine-labeled proteins, respectively
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recombinant expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D154N
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site-directed mutagenesis, the OCH mutant shows a greatly reduced value of kcat/Km derived from a very large increase in Km for the native substrate 6-oxo camphor compared to the wild-type enzyme
E244Q
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site-directed mutagenesis, the mutant shows reduced value of kcat/Km compared to the wild-type enzyme
H122A
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site-directed mutagenesis, structure analysis and comparison to the wild-type enzyme, the mutant shows a greatly reduced value of kcat, and its Km is five times that of the wild-type enzyme. The H122A mutant forms a hexamer, a dimer of trimers, identical to that of the wild-type enzyme
H145A
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site-directed mutagenesis, the OCH mutant shows a greatly reduced value of kcat/Km derived from a very large increase in Km for the native substrate 6-oxo camphor compared to the wild-type enzyme
H45A
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site-directed mutagenesis, the mutant shows reduced value of kcat/Km compared to the wild-type enzyme
D154N
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site-directed mutagenesis, the OCH mutant shows a greatly reduced value of kcat/Km derived from a very large increase in Km for the native substrate 6-oxo camphor compared to the wild-type enzyme
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E244Q
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site-directed mutagenesis, the mutant shows reduced value of kcat/Km compared to the wild-type enzyme
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H122A
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site-directed mutagenesis, structure analysis and comparison to the wild-type enzyme, the mutant shows a greatly reduced value of kcat, and its Km is five times that of the wild-type enzyme. The H122A mutant forms a hexamer, a dimer of trimers, identical to that of the wild-type enzyme
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H145A
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site-directed mutagenesis, the OCH mutant shows a greatly reduced value of kcat/Km derived from a very large increase in Km for the native substrate 6-oxo camphor compared to the wild-type enzyme
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H45A
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site-directed mutagenesis, the mutant shows reduced value of kcat/Km compared to the wild-type enzyme
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