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Information on EC 1.14.15.1 - camphor 5-monooxygenase
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1.14.15.1 camphor 5-monooxygenaseIUBMB Comments
A heme-thiolate protein (P-450). Also acts on (-)-camphor and 1,2-campholide, forming 5-exo-hydroxy-1,2-campholide.
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Word Map
- 1.14.15.1
- heme
-
ferrous
-
substrate-free
-
camphor-bound
-
substrate-bound
-
low-spin
-
high-spin
- dioxygen
-
p450terp
-
soret
- chloroperoxidase
- p450eryf
-
self-sufficient
- adrenodoxin
- aromaticivorans
-
monoxygenase
- novosphingobium
-
monooxygenation
-
ferryl
-
thiolate-ligated
-
i-helix
-
oxyferrous
- analysis
- synthesis
-
electron-electron
- biotechnology
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
+
reduced putidaredoxin
+
=
+
+
Synonyms
p450cam, cytochrome p450cam, cyp101, cytochrome p450(cam), cyp101d1, cyp101a1, camphor hydroxylase, cyp101d2, cyt p450cam, cytochrome p-450-cam, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
2-bornanone 5-exo-hydroxylase
-
-
-
-
bornanone 5-exo-hydroxylase
-
-
-
-
camphor 5-exo-hydroxylase
-
-
-
-
camphor 5-exo-methylene hydroxylase
-
-
-
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camphor 5-exohydroxylase
-
-
-
-
Camphor 5-monooxygenase
-
-
-
-
camphor hydroxylase
-
-
-
-
camphor methylene hydroxylase
-
-
-
-
cytochrome p450cam
-
-
-
-
d-camphor monooxygenase
-
-
-
-
D-camphor-exo-hydroxylase
-
-
-
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haem mono-oxygenase CYP101
-
-
-
-
methylene hydroxylase
-
-
-
-
methylene monooxygenase
-
-
-
-
oxygenase, camphor 5-mono-
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-
-
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P450cam
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-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
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-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
(+)-camphor,reduced putidaredoxin:oxygen oxidoreductase (5-hydroxylating)
A heme-thiolate protein (P-450). Also acts on (-)-camphor and 1,2-campholide, forming 5-exo-hydroxy-1,2-campholide.
CAS REGISTRY NUMBER
COMMENTARY
9030-82-4
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
3 2-methylpentane + 3 reduced ferreredoxin + O2
2-methyl-pentan-2-ol + 2-methyl-pentan-3-ol + 2-methyl-pentan-4-ol + 3 oxidized ferredoxin
-
52.5% 2-methyl-pentan-2-ol + 13% 2-methyl-pentan-3-ol, and 3% 2-methyl-pentan-4-ol for the wild-type enzyme, 5% + 12% + 30% for the mutant Y96A
-
?
adamantane + reduced ferreredoxin + O2
1-adamantol + 2-adamantol + oxidized ferredoxin + H2O
-
98% 1-adamantol + 2% 2-adamantol for the wild-type enzyme, 97% + 3% for the mutant Y96A
-
?
cyclooctane + reduced ferreredoxin + O2
cyclooctanol + cyclooctanone + oxidized ferredoxin + H2O
-
99% cyclooctanol + 1% cyclooctanone for the wild-type enzyme, 97% + 3% for the mutant Y96A
-
?
hexane + reduced ferreredoxin + O2
hexan-2-ol + hexan-3-ol + oxidized ferredoxin + H2O
-
56% hexan-2-ol + 44% hexan-3-ol for the wild-type enzyme and mutant Y96A
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
-
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
99% 5-exo-hydroxycamphor by wild-type enzyme and 92% by Y96A mutant
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
substrate of CYP101D1 and CYP101D2
-
-
?
additional information
?
-
substrate binding and activity data for wild-type CYP101D2 and variants with different substrates, gas chromatography analysis of products, overview
-
-
?
additional information
?
-
-
substrate binding and activity data for wild-type CYP101D2 and variants with different substrates, gas chromatography analysis of products, overview
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cytochrome P450
CYP101D2 is a cytochrome P450 monooxygenase
-
Ferredoxin
the CYP101D2 likely ferredoxin-binding site on the proximal face is largely positively charged, similar to that of CYP101D1
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
both the apoenzyme and the camphor-bound enzyme of CYP101D2 have open conformations with an access channel. In the active site of the camphor-bound form, the camphor carbonyl interacts with the heme-iron-bound water. The observed open structures may be conformers of the CYP101D2 enzyme that enable the substrate to enter the buried active site via a conformational selection mechanism. Two other potential camphor-binding sites exist: one located in the access channel, flanked by the B/C and F/G loops and the I helix, and the other in a cavity on the surface of the enzyme near the F helix side of the F/G loop. The second and third binding sites may be intermediate locations of substrate entry and translocation into the active site, substrate binding structure and multi-step substrate-binding mechanism, overview
additional information
role of Tyr96 in substrate binding
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). Q2G8A2_NOVAD
Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084 / F199)
417
0
46833
TrEMBL
-
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant apoenzyme and camphor-bound enzyme of CYP101D2, hanging drop vapour diffusion method, mixing of 0.001 ml of 50 mg/ml protein in 20 mM Tris-HCl, pH 8.0, and 150 mM KCl, with 0.001 ml of reservoir solution containing 0.1 M Tris/HCl, pH 8.3, 2.1 M ammonium sulfate, and 4% v/v PEG 400, equilibration against 0.2 ml reservoir solution, 18°C, 1 week, for substrate-bound form soaking of crystals in camphor-containing solution, 18°C, 1 week-1 month, X-ray diffraction structure determination and analysis at 2.4 A and 2.2. A resolution, respectively, molecular replacement and structure modeling
purified recombinant His-tagged wild-type and mutant enzymes, hanging drop vapour diffusion method, mixing of 0.001 ml of 50 mg/ml protein in crystallisation buffer containing 20 mM Tris, pH 8.0, and 150 mM KCl, with 0.001 ml of reservoir solution containing 0.1 M Tris, pH 8.3, 5% v/v PEG 400, and 1.9 M ammonium sulfate, equilivration against 0.1 ml of reservoir solution, 18°C, X-ray diffraction structure determination and analysis at 2.0 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
L253V
site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate
M98F
site-directed mutagensis, the mutant shows 85% reduced activity compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate
Y96A
site-directed mutagensis, the mutant shows increased affinity for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane, the monooxygenase activity of the mutant towards alkane substrates is enhanced compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate
additional information
additional information
substitutions at Tyr96, Met98 and Leu253 in CYP101D2 analogously to closely related CYP101A1 from Pseudomonas putida, reduce both the spin state shift on camphor binding and the camphor oxidation activity
additional information
-
substitutions at Tyr96, Met98 and Leu253 in CYP101D2 analogously to closely related CYP101A1 from Pseudomonas putida, reduce both the spin state shift on camphor binding and the camphor oxidation activity
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged CYP101D2 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, gel filtration, and anion exchange chromatography to over 95% purity
recombinant N--terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
exxpression of N-terminally His-tagged CYP101D2 in Escherichia coli strain BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Bell, S.G.; Yang, W.; Dale, A.; Zhou, W.; Wong, L.L.
Improving the affinity and activity of CYP101D2 for hydrophobic substrates
Appl. Microbiol. Biotechnol.
97
3979-3990
2013
Novosphingobium aromaticivorans (Q2G8A2), Novosphingobium aromaticivorans
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