Information on EC 1.13.12.24 - calcium-regulated photoprotein

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.13.12.24
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RECOMMENDED NAME
GeneOntology No.
calcium-regulated photoprotein
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
[aequorin] + 3 Ca2+ = [aequorin] 1,2-dioxetan-3-one
show the reaction diagram
(1c)
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-
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[aequorin] 1,2-dioxetan-3-one = [excited state blue fluorescent protein] + CO2
show the reaction diagram
(1d)
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-
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[apoaequorin containing coelenterazine] + O2 = [aequorin]
show the reaction diagram
(1b)
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[apoaequorin] + coelenterazine + O2 + 3 Ca2+ = [excited state blue fluorescent protein] + CO2
show the reaction diagram
overall reaction
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[apoaequorin] + coelenterazine = [apoaequorin containing coelenterazine]
show the reaction diagram
(1a)
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SYSTEMATIC NAME
IUBMB Comments
coelenterazine:oxygen 2-oxidoreductase (decarboxylating, calcium-dependent)
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
coeleterazine + O2
coelenteramide + CO2 + hny
show the reaction diagram
-
-
-
-
?
h-coelenterazine + O2
h-coelenteramide + CO2 + hny
show the reaction diagram
-
-
-
-
?
[aequorin] 1,2-dioxetan-3-one
[aequorin] coelenteramide + CO2 + hny
show the reaction diagram
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reaction in presence of Ca2+
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-
?
[apoaequorin] + coelenterazine + O2
[aequorin] 1,2-dioxetan-3-one
show the reaction diagram
-
-
-
-
?
[apoaequorin] + coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
show the reaction diagram
-
-
-
-
?
[apoaequorin] + coeleterazine + O2
[apoaequorin] + coelenteramide + CO2 + hny
show the reaction diagram
-
-
-
-
?
[apoaequorin] + cp-coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
show the reaction diagram
-
-
-
-
?
[apoaequorin] + f-coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
show the reaction diagram
-
-
-
-
?
[apoaequorin] + h-coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
show the reaction diagram
-
-
-
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?
[apoaequorin] + hcp-coelenterazine + O2 + 3 Ca2+
[excited state blue fluorescent protein] + CO2
show the reaction diagram
-
-
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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binding of Mg2+ to aequorin prevents the molecule from aggregating and stabilizes it in the monomeric form. Mg2+ binding induces conformational in the EF-hand loops. There are two Mg2+-binding sites, EF-hands I and III. EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and only EF-hand III seems to be occupied by Mg2+ under physiological conditions
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Barbiturate
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50 mM, complete loss of activity
Cyanate
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50 mM, complete loss of activity
glutathione
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10 mM, complete loss of activity
H2O2
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50 mM, complete loss of activity
malonate
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50 mM, complete loss of activity
NaAsO2
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20 mM, complete loss of activity
NaCl
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half-maximal inhibition was at 600 mM
nitrite
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50 mM, complete loss of activity
Thiocyanate
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50 mM, complete loss of activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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reuired. About 1% residual activity in absence of 2-mercaptoethanol
dithiothreitol
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in the presence of DTT, continuous luminescence is observed over 1 h. In the absence of DTT, the luminescence activity slowly decreases with a half period of 8.3 min
imidazole
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luminescence activity of BFP-aq 1s stimulated 1.5- to 2fold by imidazole at concentrations of 30-300 mM. Imidazole has no effect on the binding affinity of coelenterazine and may act as a catalytic base
additional information
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1H-pyrazole, 2-methylimidazole, 4-methylimidazole, histamine, L-histidine, urocanic acid, and 5-amino-4-imidazole carboxamide do not act as stimulators at concentrations of 30 and 150 mM
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.1 - 8.3
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21400
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calaculated from amino acid sequence
23000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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x * 21400, calculated
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
to 1.7 A resolution. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-protein retains the same compact scaffold and overall fold as the unreacted photoprotein containing the bound substrate, 2-hydroperoxycoelenterazine
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to 2.3 A resolution. Aequorin is a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide
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to 1.7 A resolution. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-protein retains the same compact scaffold and overall fold as the unreacted photoprotein containing the bound substrate, 2-hydroperoxycoelenterazine and also the same as the Ca2+-discharged obelin bound with the product, coelenteramide
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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unstable below
745437
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28.8
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Cys-free mutant, melting temperature
44
Q6J4T7
wild-type, 50% inactivation of aequorin bioluminescence by a 30-min heat shock
44.6
Cys-free mutant, melting temperature
48
Q6J4T7
mutant L170I, 50% inactivation of aequorin bioluminescence by a 30-min heat shock
49.1
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wild-type, melting temperature
53
Q6J4T7
mutant Q168R, 50% inactivation of aequorin bioluminescence by a 30-min heat shock
53.3
wild-type, melting temperature
57
Q6J4T7
mutant Q168R/L170I, 50% inactivation of aequorin bioluminescence by a 30-min heat shock
95
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3 min, complete loss of activity. Fluorescence recovers to over 93% after incubating for a further 20 min at 24C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
aequorin is extremely sensitive to presence of Ca2+, the solutions usually faintly luminescing spontaneously, resulting in a gradual loss of capacity to luminesce. Spontaneous luminescence, with the accompanying decrease in activity, can be eliminated by saturating the aequorin plus EDTA solutions with (NH4)2SO4 and keeping at -20
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protein is relatively stable in 0.01 M Na2EDTA
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
benzyl alcohol
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50 mM, 10% increase in activity
n-amyl alcohol
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50 mM, 7% increase in activity
n-Butanol
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50 mM, 8% increase in activity
n-heptanol
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50 mM, 30% increase in activity
n-hexanol
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50 mM, 10% increase in activity
n-octanol
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50 mM, 18% increase in activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
upon treatment with urea and 2-mercaptoethanol, a blue fluorescent compound (AF-350), and a protein (apoaequorin-SH) are obtained
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Saccharomyces cerevisiae and Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A123D
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mutant shows Ca2+-dependent chemiluminescence
C145S
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61% of wild-type activity
C145S/C152S
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1% of wild-type activity
C145S/C152S/C180S
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21% of wild-type activity. Contrary to wild-type, 49% residual activity in absence of 2-mercaptoethanol. The regeneration of the triple mutant aequorin is sharply inhibited by 2-mercaptoethanol
C145S/C180S
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68% of wild-type activity. Contrary to wild-type, 28% residual activity in absence of 2-mercaptoethanol
C152S
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46% of wild-type activity
C152S/C180S
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17% of wild-type activity. Contrary to wild-type, 14% residual activity in absence of 2-mercaptoethanol
C180S
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13% of wild-type activity
D119A
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mutant is an active photoprotein, Ca2+ affinity is reduced by a factor of 20 compared to the wild type
F149
Q6J4T7
residue participates in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues
H169A
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1.1% of wild-type activity
H169F
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modification leads to complete loss of activity
H169W
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modification leads to complete loss of activity
H16A
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26% of wild-type activity
H16F
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0.1% of wild-type activity
H18A
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20% of wild-type activity
H27A
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70% of wild-type activity
H27N
Q6J4T7
18fold increase of wild-type activity
H27N/Q168R
Q6J4T7
13fold increase of wild-type activity
H27P
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67% of wild-type activity
H58F
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1.4% of wild-type activity
H58Y
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15% of wild-type activity
K17R
Q6J4T7
13fold increase of wild-type activity
K17R/H27N/Q168R
Q6J4T7
11fold increase of wild-type activity
K17R/L170I
Q6J4T7
8fold increase of wild-type activity
K17R/N26D
Q6J4T7
14fold increase of wild-type activity
K17R/N26D/Q168R
Q6J4T7
14fold increase of wild-type activity
K17R/Q168R
Q6J4T7
25fold increase of wild-type activity
K30E
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mutant shows Ca2+-dependent chemiluminescence
L170I
Q6J4T7
16fold increase of wild-type activity, an increase of the photoprotein lifetime at 37C and increased thermostability
N26D
Q6J4T7
13fold increase of wild-type activity
N26D/Q168R
Q6J4T7
8fold increase of wild-type activity
Q168
Q6J4T7
residue participates in stabilization of the coelenterazine peroxide and the triggering of photon emission by linking the third EF-hand to Trp-129 and His-169 coelenterazine binding residues
Q168R
Q6J4T7
11fold increase of wild-type activity, increase of the photoprotein lifetime at 37C and increased thermostablility
Q168R/L170I
Q6J4T7
mutant displays increased thermostablility
V25A
Q6J4T7
10fold increase of wild-type activity
V25I/L170I
Q6J4T7
24fold increase of wild-type activity
V25I/Q168R
Q6J4T7
20fold increase of wild-type activity
Y82F
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mutation leads to removal of a H-bond from Tyr82 to the bound coelenteramide, and shifts its bioluminescence from 469 nm for wild-type to 501 nm. Mutant is stable with good activity and expressible in mammalian cells
F88H
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mutation shifts the bioluminescence maximum from 482 nm for wild-type to 459 nm. 105% of wild-type bioluminescence yield
F88R
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mutation shifts the bioluminescence maximum from 482 nm for wild-type to 474 nm. 99% of wild-type bioluminescence yield
F88W
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mutation shifts the bioluminescence maximum from 482 nm for wild-type to 477 nm. 100% of wild-type bioluminescence yield
F88Y
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mutation shifts the bioluminescence maximum from 482 nm for wild-type to 453 nm. 100% of wild-type bioluminescence yield
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
synthesis