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Information on EC 1.14.14.133 - 1,8-cineole 2-endo-monooxygenase and Organism(s) Citrobacter braakii and UniProt Accession Q8VQF6
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1.14.14.133 1,8-cineole 2-endo-monooxygenaseIUBMB Comments
A cytochrome P-450 (heme-thiolate) protein that 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.
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Word Map
- 1.14.14.133
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p450cam
- heme
- monooxygenases
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h-bonding
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citrobacter
- monoterpenes
- fmn
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substrate-bound
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fmn-containing
- braakii
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substrate-free
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monoterpenoid
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sesquiterpene
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s-oxidations
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bicyclic
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terpene
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ferryl
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low-spin
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regioselective
The taxonomic range for the selected organisms is: Citrobacter braakii
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Synonyms
p450cin, cyp176a1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CYP176A
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CYP176A1
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P450cin
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PATHWAY SOURCE
PATHWAYS
SYSTEMATIC NAME
IUBMB Comments
1,8-cineole,[reduced flavodoxin]:oxygen oxidoreductase (2-endo-hydroxylating)
A cytochrome P-450 (heme-thiolate) protein that 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.
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
1,8-cineole + [reduced flavodoxin] + O2
(1R)-6beta-hydroxycineole + [oxidized flavodoxin] + H2O
1,8-cineole + [reduced flavodoxin] + O2
6beta-hydroxycineole + [oxidized flavodoxin] + H2O
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-
-
?
1,8-cineole + [reduced NADPH-hemoprotein reductase] + O2
2-endo-hydroxy-1,8-cineole + [oxidized NADPH-hemoprotein reductase] + H2O
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?
2,2-dimethylbicyclo[2.2.2]octane + [reduced flavodoxin] + O2
? + [oxidized flavodoxin] + H2O
i.e. cinane
identification of least seven oxidised derivatives
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?
camphane + [reduced flavodoxin] + O2
camphor + [oxidized flavodoxin] + H2O
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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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?
1,8-cineole + [reduced flavodoxin] + O2
(1R)-6beta-hydroxycineole + [oxidized flavodoxin] + H2O
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?
1,8-cineole + [reduced flavodoxin] + O2
(1R)-6beta-hydroxycineole + [oxidized flavodoxin] + H2O
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single product
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?
1,8-cineole + [reduced flavodoxin] + O2
(1R)-6beta-hydroxycineole + [oxidized flavodoxin] + H2O
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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?
additional information
?
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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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additional information
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P450cin catalyzes the stereoselective hydoxylation of 1,8-cineole to 2beta-hydroxy-1,8-cineole. The two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
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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
1,8-cineole + [reduced NADPH-hemoprotein reductase] + O2
2-endo-hydroxy-1,8-cineole + [oxidized NADPH-hemoprotein reductase] + H2O
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?
additional information
?
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P450cin catalyzes the stereoselective hydoxylation of 1,8-cineole to 2beta-hydroxy-1,8-cineole. The two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADPH-hemoprotein reductase
A flavoprotein containing both FMN and FAD. This enzyme catalyses the transfer of electrons from NADPH, an obligatory two-electron donor, to microsomal P-450 monooxygenases, EC 1.14.14._
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FMN
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
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
large spin state change of the heme iron associated with binding of cineole
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cindoxin
Cdx, UniProt ID Q8VQF4, the FMN-containing redox partner to P450cin. Brownian dynamics-molecular dynamics docking method is used to produce a model of Cdx with its redox partner, enzyme P450cin, overview. Potential importance of Cdx Tyr96 in bridging the FMN and heme cofactors as well P450cin Arg102 and Arg346. Arg346 plays an important role in electron transfer. Arg102 also interacts with a P450cin heme propionate. Redox partner binding stabilizes the open low-spin conformation of P450cam and greatly decreases the stability of the oxy complex. Crystal structure determination of wild-type and mutant cindoxins
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
Michaelis-Menten kinetics
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additional information
additional information
stopped-flow oxyP450cin formation and P450cin-Cdx electron transfer analysis
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
44
[reduced NADPH-hemoprotein reductase]
pH 7.5, 22°C, P450cin mutant R346A
82.4
[reduced NADPH-hemoprotein reductase]
pH 7.5, 22°C, P450cin mutant R102A
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
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
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
P450cin from Citrobacter braakii is a three-component class I P450 system except that its flavin-containing components resemble class II P450s
additional information
additional information
redox partner binding stabilizes the open low-spin conformation of P450cam and greatly decreases the stability of the oxy complex
additional information
the two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
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). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
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
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
N242A
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
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
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
Q385H/V386S/T77N/L88R
directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems, for P450 monooxygenase P450cin: directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III) sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of sequence saturation mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generate a variant Q385H/V386S/T77N/L88R, named KB8, with a 3.8fold increase in catalytic efficiency (0.028 mM/min) compared to P450cin-ADD-CinC (0.007 mM/min). Mutant variant KB8 exhibits a 1.5fold higher product formation compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as cofactor and 4fold higher product formation rate than the P450cin-ADD-CinC mutant. Molecular docking of CoIIIsep into P450cin fusion protein
R102A
site-directed mutagenesis, the mutant is unable to bind the redox partner cindoxin and shows only 5% of wild-type enzyme NADPH turnover
R346A
site-directed mutagenesis, the mutant is unable to bind the redox partner cindoxin and shows only 10% of wild-type enzyme NADPH turnover
T243A
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
additional information
construction of a functional P450 CinA-(heme center)-CinC (reductase) fusion protein separated by a linker of 10 amino acid in length, here named as P450cin-ADDCinC, to replace the multi-component system in the hydroxylation of 1,8-cineole. The P450cin-ADD-CinC variant able to hydroxylate 1,8-cineole to 2-beta-hydroxy-1,8-cineole using the alternative electron delivery systems in which zinc dust or a platinum electrode substitute the NADPH as electron source while CoIIIsep acts as electron mediator. Generation of random mutagenesis libraries and expression of mutant libraries
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of enzyme mutants in Escherichia coli strain BL21 Gold (DE3) lacIQ
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Slessor, K.E.; Farlow, A.J.; Cavaignac, S.M.; Stok, J.E.; De Voss, J.J.
Oxygen activation by P450(cin): Protein and substrate mutagenesis
Arch. Biochem. Biophys.
507
154-162
2011
Citrobacter braakii (Q8VQF6)
Meharenna, Y.T.; Li, H.; Hawkes, D.B.; Pearson, A.G.; De Voss, J.; Poulos, T.L.
Crystal structure of P450cin in a complex with its substrate, 1,8-cineole, a close structural homologue to D-camphor, the substrate for P450cam
Biochemistry
43
9487-9494
2004
Citrobacter braakii (Q8VQF6)
Hawkes, D.B.; Slessor, K.E.; Bernhardt, P.V.; De Voss, J.J.
Cloning, expression and purification of cindoxin, an unusual Fmn-containing cytochrome p450 redox partner
ChemBioChem
11
1107-1114
2010
Citrobacter braakii (Q8VQF6)
Hawkes, D.B.; Adams, G.W.; Burlingame, A.L.; Ortiz de Montellano, P.R.; De Voss, J.J.
Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization
J. Biol. Chem.
277
27725-27732
2002
Citrobacter braakii (Q8VQF6)
Kimmich, N.; Das, A.; Sevrioukova, I.; Meharenna, Y.; Sligar, S.G.; Poulos, T.L.
Electron transfer between cytochrome P450cin and its FMN-containing redox partner, cindoxin
J. Biol. Chem.
282
27006-27011
2007
Citrobacter braakii (Q8VQF6)
Meharenna, Y.T.; Slessor, K.E.; Cavaignac, S.M.; Poulos, T.L.; De Voss, J.J.
The critical role of substrate-protein hydrogen bonding in the control of regioselective hydroxylation in p450cin
J. Biol. Chem.
283
10804-10812
2008
Citrobacter braakii (Q8VQF6), Citrobacter braakii
Madrona, Y.; Hollingsworth, S.; Tripathi, S.; Fields, J.; Rwigema, J.; Tobias, D.; Poulos, T.
Crystal structure of cindoxin, the P450cin redox partner
Biochemistry
53
1435-1446
2014
Citrobacter braakii (Q8VQF6)
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