Information on EC 1.14.13.156 - 1,8-cineole 2-endo-monooxygenase

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The expected taxonomic range for this enzyme is: Citrobacter braakii

EC NUMBER
COMMENTARY
1.14.13.156
-
RECOMMENDED NAME
GeneOntology No.
1,8-cineole 2-endo-monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
1,8-cineole + NADPH + H+ + O2 = 2-endo-hydroxy-1,8-cineole + NADP+ + H2O
show the reaction diagram
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PATHWAY
KEGG Link
MetaCyc Link
Monoterpenoid biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
1,8-cineole,NADPH:oxygen oxidoreductase (2-endo-hydroxylating)
A heme-thiolate protein (P-450) which uses a flavodoxin-like redox partner to reduce the heme iron. Isolated from the bacterium Citrobacter braakii, which can use 1,8-cineole as the sole source of carbon.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
CYP176A
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-
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CYP176A
Q8VQF6
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CYP176A1
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CYP176A1
Q8VQF6
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P450cin
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-
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ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
CinA, part of putative operon consisting of three open reading frames. CinB and cinC appear to encode the expected redox partners for a catalytically functional P450 system
UniProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,8-cineole + NADPH + H+ + O2
(1R)-6beta-hydroxycineole + NADP+ + H2O
show the reaction diagram
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-
-
-
?
1,8-cineole + NADPH + H+ + O2
(1R)-6beta-hydroxycineole + NADP+ + H2O
show the reaction diagram
Q8VQF6
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single product
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?
1,8-cineole + NADPH + H+ + O2
(1R)-6beta-hydroxycineole + NADP+ + H2O
show the reaction diagram
Q8VQF6
enzyme displays a high affinity for cineole 1 with KD 0.7 microM, and a large spin state change of the heme iron associated with binding of cineole
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?
1,8-cineole + NADPH + H+ + O2
6beta-hydroxycineole + NADP+ + H2O
show the reaction diagram
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-
-
-
?
2,2-dimethylbicyclo[2.2.2]octane + NADPH + H+ + O2
? + NADP+ + H2O
show the reaction diagram
Q8VQF6
i.e. cinane
identification of least seven oxidised derivatives
-
?
camphane + NADPH + H+ + O2
camphor + NADP+ + H2O
show the reaction diagram
Q8VQF6
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main product in presence of excess NADPH, plus epi-camphor at a rate of 3:1, and several minor products
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?
additional information
?
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Q8VQF6
a hydroxyl group on the substrate is vital, and in its absence catalytic turnover is effectively abolished. In the absence of the ethereal oxygen there is still a significant amount of coupling of the NADPH-reducing equivalents to the formation of oxidised product. The substrate itself is not important in controlling oxygen activation, but is essential for regio- and stereoselective substrate oxidation
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COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
FMN
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redox partner is cindoxin, containing FMN. Cindoxin might be different to other flavodoxins that interact with P450s, as both redox states of cindoxin could be catalytically relevant. Cindoxin supports regio- and stereoselective P450cin-catalysed cineole oxidation to (1R)-6beta-hydroxycineole with turnover rates up to 1500 per min
FMN
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the FAD/FMN reductase consists of two separate polypeptides where the FMN protein, cindoxin, shuttles electrons between the FAD-containing cindoxin reductase and P450cin. Reaction is highly ionic strength-dependent. The fully reduced hydroquinone is the electron-donating species. Surface interactions are rather different from other P450 proteins
heme
Q8VQF6
large spin state change of the heme iron associated with binding of cineole
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
Q8VQF6
x * 45000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
in complex with substrate 1,8-cineole, to 1.7 A resolution, and comparison with P450cam, EC 1.14.15.1. The active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s, is replaced by an ordered loop that results in substantial changes in active site topography. Cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe2S2 redox partner used by cytochrome P450cam
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X-ray crystal structures of the substrate-free and -bound N242A mutant to 2.0 and 3.0 A resolution, respcetively. Mutation results in a reorientation of the substrate such that (R)-6'-hydroxycineole should be a major product
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Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression in Escherichia coli
Q8VQF6
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
N242A
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no change in characteristic CO-bound spectrum and spectrally determined KD for substrate binding. Mutation leads to modest effects on enzyme activity and on the diversion of the NADPH-reducing equivalent toward unproductive peroxide formation, but results in a reorientation of the substrate such that (R)-6'-hydroxycineole is a major product
N242T
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significant drop in the rate of NADPH consumption. In addition to wild-type product (1R)-6beta-hydroxycineole, products (1R)-6alpha-hydroxycineole 2b and (1S)-6alpha-hydroxycineole are formed at 22% and 31%, respectively
N242T/T243A
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significant drop in the rate of NADPH consumption. In addition to wild-type product (1R)-6beta-hydroxycineole, products (1R)-6alpha-hydroxycineole 2b and (1S)-6alpha-hydroxycineole are formed at 18% and 39%, respectively
T243A
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increase in the rate of NADPH consumption of 30%. Like in wild-type, single product is (1R)-6beta-hydroxycineole. T243 is not involved in controlling the protonation of the hydroperoxy species