BRENDA - Enzyme Database show
show all sequences of 1.13.11.71

Substrate specificity of purified recombinant chicken beta-carotene 9',10'-oxygenase (BCO2)

Dela Sena, C.; Sun, J.; Narayanasamy, S.; Riedl, K.M.; Yuan, Y.; Curley, R.W.; Schwartz, S.J.; Harrison, E.H.; J. Biol. Chem. 291, 14609-14619 (2016)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
gene BCO2, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21-Gold (DE3)
Gallus gallus
gene BCO2, sequence comparisons, recombinant expression of His-tagged, catalytically inactive enzyme BCO2 in Escherichia coli strain BL21-Gold (DE3), mostly in inclusion bodies
Homo sapiens
Localization
Localization
Commentary
Organism
GeneOntology No.
Textmining
mitochondrion
-
Gallus gallus
5739
-
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
all-trans-beta-carotene + O2
Gallus gallus
-
all-trans-10'-apo-beta-carotenal + beta-ionone
-
-
?
all-trans-beta-carotene + O2
Homo sapiens
-
all-trans-10'-apo-beta-carotenal + beta-ionone
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Gallus gallus
E1C8E0
-
-
Homo sapiens
B2RCV8
-
-
Purification (Commentary)
Commentary
Organism
a small amount of recombinant, catalytically inactive His-tagged enzyme from Escherichia coli strain BL21-Gold (DE3) by cobalt affinity chromatography
Homo sapiens
recombinant His-tagged enzyme from Escherichia coli strain BL21-Gold (DE3) by cobalt affinity chromatography
Gallus gallus
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
9-cis-beta-carotene + O2
-
745345
Gallus gallus
9-cis-beta-apo-10'-carotenal + beta-ionone
-
-
-
?
all-trans-beta-carotene + O2
-
745345
Gallus gallus
all-trans-10'-apo-beta-carotenal + beta-ionone
-
-
-
?
all-trans-beta-carotene + O2
-
745345
Homo sapiens
all-trans-10'-apo-beta-carotenal + beta-ionone
-
-
-
?
alpha-carotene + O2
-
745345
Gallus gallus
beta-apo-10'-carotenal + alpha-apo-10'-carotenal
-
-
-
?
beta-cryptoxanthin + O2
-
745345
Gallus gallus
3-hydroxy-beta-apo-10'-carotenal + beta-ionone
-
-
-
?
beta-cryptoxanthin + O2
-
745345
Gallus gallus
3-hydroxy-beta-apo-8'-carotenal + beta-cyclocitral
-
-
-
?
beta-cryptoxanthin + O2
-
745345
Gallus gallus
beta-apo-10'-carotenal + 3-hydroxy-beta-ionone
-
-
-
?
lutein + O2
-
745345
Gallus gallus
3-hydroxy-alpha-apo-10'-carotenal + 3-hydroxy-beta-ionone
-
-
-
?
lutein + O2
-
745345
Gallus gallus
3-hydroxy-beta-apo-10'-carotenal + 3-hydroxy-alpha-ionone
-
-
-
?
additional information
like BCO1, EC 1.13.11.63, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids beta-carotene, alpha-carotene, and beta-cryptoxanthin. Its catalytic activity with beta-carotene as substrate is at least 10fold lower than that of BCO1. Purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-beta-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and beta-apo-carotenoids. Apo-10'-carotenoids are detected as enzymatic products by HPLC, and the identities are confirmed by LC-MS. Small amounts of 3-hydroxy-beta-apo-8'-carotenal are also consistently detected in BCO2-beta-cryptoxanthin reaction mixtures. With the exception of this activity with beta-cryptoxanthin, BCO2 cleaves specifically at the 9'-10'-bond to produce apo-10'-carotenoids. BCO2 is regioselective (or even regiospecific) for the trans 9'-10'-end of 9-cis-beta-carotene due to the size of the substrate tunnel. Unlike murine BCO2, chicken BCO2 cannot perform a second cleavage on beta-apo-10'-carotenal to yield rosafluene dialdehyde, and it has no activity with all-trans-lycopene, while it cleaves cis-lycopene isomers. Chicken BCO2 also does not have activity with the other beta-apocarotenals in the series (beta-apo-8'-carotenal, beta-apo-12'-carotenal, and beta-apo-14'-carotenal). Importance of the 3-hydroxy-beta-ionone ring for BCO2 cleavage, preference of BCO2 for substrates that contain a 3-hydroxy-beta-ionone ring
745345
Gallus gallus
?
-
-
-
-
zeaxanthin + O2
-
745345
Gallus gallus
3-hydroxy-beta-apo-10'-carotenal + 3-hydroxy-beta-ionone
-
-
-
?
Temperature Optimum [C]
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
37
-
assay at
Gallus gallus
37
-
assay at
Homo sapiens
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Gallus gallus
8
-
assay at
Homo sapiens
Cloned(Commentary) (protein specific)
Commentary
Organism
gene BCO2, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21-Gold (DE3)
Gallus gallus
gene BCO2, sequence comparisons, recombinant expression of His-tagged, catalytically inactive enzyme BCO2 in Escherichia coli strain BL21-Gold (DE3), mostly in inclusion bodies
Homo sapiens
Localization (protein specific)
Localization
Commentary
Organism
GeneOntology No.
Textmining
mitochondrion
-
Gallus gallus
5739
-
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
all-trans-beta-carotene + O2
Gallus gallus
-
all-trans-10'-apo-beta-carotenal + beta-ionone
-
-
?
all-trans-beta-carotene + O2
Homo sapiens
-
all-trans-10'-apo-beta-carotenal + beta-ionone
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
a small amount of recombinant, catalytically inactive His-tagged enzyme from Escherichia coli strain BL21-Gold (DE3) by cobalt affinity chromatography
Homo sapiens
recombinant His-tagged enzyme from Escherichia coli strain BL21-Gold (DE3) by cobalt affinity chromatography
Gallus gallus
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
9-cis-beta-carotene + O2
-
745345
Gallus gallus
9-cis-beta-apo-10'-carotenal + beta-ionone
-
-
-
?
all-trans-beta-carotene + O2
-
745345
Gallus gallus
all-trans-10'-apo-beta-carotenal + beta-ionone
-
-
-
?
all-trans-beta-carotene + O2
-
745345
Homo sapiens
all-trans-10'-apo-beta-carotenal + beta-ionone
-
-
-
?
alpha-carotene + O2
-
745345
Gallus gallus
beta-apo-10'-carotenal + alpha-apo-10'-carotenal
-
-
-
?
beta-cryptoxanthin + O2
-
745345
Gallus gallus
3-hydroxy-beta-apo-10'-carotenal + beta-ionone
-
-
-
?
beta-cryptoxanthin + O2
-
745345
Gallus gallus
3-hydroxy-beta-apo-8'-carotenal + beta-cyclocitral
-
-
-
?
beta-cryptoxanthin + O2
-
745345
Gallus gallus
beta-apo-10'-carotenal + 3-hydroxy-beta-ionone
-
-
-
?
lutein + O2
-
745345
Gallus gallus
3-hydroxy-alpha-apo-10'-carotenal + 3-hydroxy-beta-ionone
-
-
-
?
lutein + O2
-
745345
Gallus gallus
3-hydroxy-beta-apo-10'-carotenal + 3-hydroxy-alpha-ionone
-
-
-
?
additional information
like BCO1, EC 1.13.11.63, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids beta-carotene, alpha-carotene, and beta-cryptoxanthin. Its catalytic activity with beta-carotene as substrate is at least 10fold lower than that of BCO1. Purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-beta-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and beta-apo-carotenoids. Apo-10'-carotenoids are detected as enzymatic products by HPLC, and the identities are confirmed by LC-MS. Small amounts of 3-hydroxy-beta-apo-8'-carotenal are also consistently detected in BCO2-beta-cryptoxanthin reaction mixtures. With the exception of this activity with beta-cryptoxanthin, BCO2 cleaves specifically at the 9'-10'-bond to produce apo-10'-carotenoids. BCO2 is regioselective (or even regiospecific) for the trans 9'-10'-end of 9-cis-beta-carotene due to the size of the substrate tunnel. Unlike murine BCO2, chicken BCO2 cannot perform a second cleavage on beta-apo-10'-carotenal to yield rosafluene dialdehyde, and it has no activity with all-trans-lycopene, while it cleaves cis-lycopene isomers. Chicken BCO2 also does not have activity with the other beta-apocarotenals in the series (beta-apo-8'-carotenal, beta-apo-12'-carotenal, and beta-apo-14'-carotenal). Importance of the 3-hydroxy-beta-ionone ring for BCO2 cleavage, preference of BCO2 for substrates that contain a 3-hydroxy-beta-ionone ring
745345
Gallus gallus
?
-
-
-
-
zeaxanthin + O2
-
745345
Gallus gallus
3-hydroxy-beta-apo-10'-carotenal + 3-hydroxy-beta-ionone
-
-
-
?
Temperature Optimum [C] (protein specific)
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
37
-
assay at
Gallus gallus
37
-
assay at
Homo sapiens
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Gallus gallus
8
-
assay at
Homo sapiens
General Information
General Information
Commentary
Organism
metabolism
the substrates that are inactive with BCO2 (all-trans-lycopene and beta-apocarotenals) are substrates for BCO1
Gallus gallus
physiological function
beta-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10'-bond to yield an ionone and an apo-10'-carotenoid. Chicken BCO2 has broader substrate specificity than BCO1, consistent with its proposed function of preventing oxidative stress brought about by carotenoid accumulation in the mitochondria. It cleaves only full-length carotenoids with ionone rings, and the hydroxylated carotenoids are cleaved to a greater extent than their hydrocarbon counterparts under the conditions tested
Gallus gallus
physiological function
beta-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10'-bond to yield an ionone and an apo-10'-carotenoid
Homo sapiens
General Information (protein specific)
General Information
Commentary
Organism
metabolism
the substrates that are inactive with BCO2 (all-trans-lycopene and beta-apocarotenals) are substrates for BCO1
Gallus gallus
physiological function
beta-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10'-bond to yield an ionone and an apo-10'-carotenoid. Chicken BCO2 has broader substrate specificity than BCO1, consistent with its proposed function of preventing oxidative stress brought about by carotenoid accumulation in the mitochondria. It cleaves only full-length carotenoids with ionone rings, and the hydroxylated carotenoids are cleaved to a greater extent than their hydrocarbon counterparts under the conditions tested
Gallus gallus
physiological function
beta-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10'-bond to yield an ionone and an apo-10'-carotenoid
Homo sapiens
Other publictions for EC 1.13.11.71
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [C]
Temperature Range [C]
Temperature Stability [C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [C] (protein specific)
Temperature Range [C] (protein specific)
Temperature Stability [C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
745596
Guo
Ablation of beta,beta-caroten ...
Mus musculus
J. Nutr. Biochem.
46
74-82
2017
-
-
-
-
-
-
-
-
1
-
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1
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1
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2
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1
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1
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1
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2
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1
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2
2
-
-
-
745345
Dela Sena
Substrate specificity of puri ...
Gallus gallus, Homo sapiens
J. Biol. Chem.
291
14609-14619
2016
-
-
2
-
-
-
-
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1
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2
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2
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2
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11
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2
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2
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2
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1
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2
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2
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11
-
2
-
-
-
2
-
-
-
-
3
3
-
-
-
745753
Gong
Mitochondrial beta-carotene 9 ...
Homo sapiens
Mol. Cancer Res.
14
966-975
2016
-
-
-
-
-
-
-
-
1
-
-
1
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1
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8
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1
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1
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1
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8
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1
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-
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-
2
3
3
2
-
-
744160
Kim
Tissue- and sex-specific effe ...
Mus musculus, Mus musculus C57BL/6
Arch. Biochem. Biophys.
572
11-18
2015
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-
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2
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2
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10
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2
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10
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2
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744161
Raghuvanshi
Cellular localization of ?-ca ...
Rattus norvegicus
Arch. Biochem. Biophys.
572
19-27
2015
-
-
-
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-
-
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2
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1
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1
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4
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1
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2
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1
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4
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1
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1
1
-
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744574
Wei
Purification and characteriza ...
Saccharomyces cerevisiae, Saccharomyces cerevisiae ULI3
Biotechnol. Lett.
37
1993-1998
2015
3
1
-
-
-
-
5
3
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5
-
8
8
4
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1
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1
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10
1
1
1
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3
1
1
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3
1
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5
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3
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5
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8
8
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1
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1
-
10
1
1
1
-
3
1
1
-
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-
1
1
-
3
3
744613
Jlali
Nutrigenetics of carotenoid m ...
Gallus gallus
Br. J. Nutr.
111
2079-2088
2014
-
-
1
-
1
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-
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-
1
-
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3
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1
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3
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746290
Li
Inactivity of human beta,beta ...
Homo sapiens, Mus musculus, Mus musculus C57/BL6
Proc. Natl. Acad. Sci. USA
111
10173-10178
2014
-
-
2
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2
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10
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10
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2
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11
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2
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2
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10
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2
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11
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2
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2
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5
5
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727965
Amengual
Two carotenoid-oxygenases cont ...
Mus musculus
J. Biol. Chem.
288
34081-34096
2013
-
-
1
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1
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1
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1
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1
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1
1
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728057
Liu
Carotenoid cleavage dioxygenas ...
Lotus japonicus
J. Exp. Bot.
64
1967-1981
2013
-
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1
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1
1
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727353
Lobo
BCDO2 acts as a carotenoid sca ...
Danio rerio, Homo sapiens
Development
139
2966-2977
2012
-
-
-
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1
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3
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6
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2
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3
3
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719151
Kim
Production of beta-apo-10'-car ...
Homo sapiens
Biotechnol. Lett.
33
1195-1200
2011
-
1
1
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1
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1
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1
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1
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1
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5
1
1
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1
1
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1
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1
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1
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1
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5
1
1
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1
1
719438
Amengual
A mitochondrial enzyme degrade ...
Mus musculus
FASEB J.
25
948-959
2011
-
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1
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-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
728552
Amengual
Beta-carotene reduces body adi ...
Mus musculus
PLoS ONE
6
e20644
2011
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
-
-
-
-
-
-
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-
-
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-
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-
-
-
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-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
714616
Vage
A nonsense mutation in the bet ...
Ovis aries
BMC Genet.
11
10
2010
-
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
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1
-
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-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
720559
Ledger
Modified CAROTENOID CLEAVAGE D ...
Actinidia chinensis
New Phytol.
188
803-813
2010
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
720702
Auldridge
Characterization of three memb ...
Arabidopsis thaliana
Plant J.
45
982-993
2006
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
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-
-
-
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-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
720152
Lindqvist
Cell type-specific expression ...
Homo sapiens
J. Histochem. Cytochem.
53
1403-1412
2005
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
16
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
16
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
719823
Schwartz
The biochemical characterizati ...
Arabidopsis thaliana
J. Biol. Chem.
279
46940-46945
2004
1
-
-
-
1
-
-
1
1
-
-
-
1
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
1
-
-
-
-
1
1
-
-
-
1
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
719806
Kiefer
Identification and characteriz ...
Homo sapiens, Mus musculus
J. Biol. Chem.
276
14110-14116
2001
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
8
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
8
-
-
3
-
-
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-
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-