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Information on EC 1.13.12.5 - Renilla-type luciferase
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1.13.12.5 Renilla-type luciferaseIUBMB Comments
This enzyme has been studied from the soft coral Renilla reniformis. Before the reaction occurs the substrate is sequestered by a coelenterazine-binding protein. Elevation in the concentration of calcium ions releases the substrate, which then interacts with the luciferase. Upon binding the substrate, the enzyme catalyses an oxygenation, producing a very short-lived hydroperoxide that cyclizes into a dioxetanone structure, which collapses, releasing a CO2 molecule. The spontaneous breakdown of the dioxetanone releases the energy (about 50 kcal/mole) that is necessary to generate the excited state of the coelenteramide product, which is the singlet form of the monoanion. In vivo the product undergoes the process of nonradiative energy transfer to an accessory protein, a green fluorescent protein (GFP), which results in green bioluminescence. In vitro, in the absence of GFP, the product emits blue light.
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- 1.13.12.5
-
bioluminescence
-
luminescence
-
photoproteins
- firefly
- agonist
- ventricular
-
contractile
-
tension
- ferret
-
emit
-
twitch
-
isometric
-
papillary
-
depolarization
-
ca2+-sensitive
-
microinjected
- myocardium
-
sarcoplasmic
- aequorea
- caffeine
-
ryanodine
- myofilaments
-
inotropic
-
jellyfish
-
calcium-sensitive
- luciferases
- victoria
-
excitation-contraction
-
fura-2
-
replicons
-
ca2+cyt
-
tetanic
-
light-emitting
- analysis
-
glow
- trabeculae
- insp3
- biotechnology
- photinus
-
signal-to-background
-
ca2+-regulated
-
ca2+-triggered
- barnacle
-
luminometer
-
luminescence-based
-
myoplasmic
-
ca2+-mobilizing
- molecular biology
- diagnostics
- pharmacology
-
isovolumic
-
charge-coupled
-
luciferase-based
- pyralis
-
photomultiplier
The enzyme appears in viruses and cellular organisms
Reaction Schemes
Synonyms
aequorin, renilla luciferase, gaussia luciferase, gaussia princeps luciferase, renilla reniformis luciferase, rluc8, clytin, oplophorus luciferase, bfp-aq, gaussia-luciferase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
aequorin
BFP-aq
blue fluorescent protein from the calcium-binding photoprotein aequorin
-
a complex of Ca2+-bound apoaequorin and coelenteramide
firefly luciferase
fluc
Gaussia luciferase
GFP-aq
-
a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1
GLase
luciferase
luciferase (Renilla luciferin)
-
-
-
-
Renilla luciferase
Renilla luciferin 2-monooxygenase
-
-
-
-
Renilla-luciferin 2-monooxygenase
Renilla-type luciferase
-
-
-
-
RLuc
aequorin
-
-
-
-
Renilla luciferase
-
Renilla-luciferin 2-monooxygenase
-
-
-
-
RLuc
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
coelenterazine h + O2 = coelenterazine h dioxetanone
(1a)
-
-
-
coelenterazine h dioxetanone = excited coelenteramide h monoanion + CO2
(1b)
-
-
-
coelenterazine h + O2 = excited coelenteramide h monoanion + CO2
mechanism
-
coelenterazine h + O2 = excited coelenteramide h monoanion + CO2
mechanism
-
coelenterazine h + O2 = excited coelenteramide h monoanion + CO2
mechanism
-
coelenterazine h + O2 = excited coelenteramide h monoanion + CO2
overall reaction
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
-
-
-
-
oxidation
-
-
-
-
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SYSTEMATIC NAME
IUBMB Comments
coelenterazine h:oxygen 2-oxidoreductase (decarboxylating)
This enzyme has been studied from the soft coral Renilla reniformis. Before the reaction occurs the substrate is sequestered by a coelenterazine-binding protein. Elevation in the concentration of calcium ions releases the substrate, which then interacts with the luciferase. Upon binding the substrate, the enzyme catalyses an oxygenation, producing a very short-lived hydroperoxide that cyclizes into a dioxetanone structure, which collapses, releasing a CO2 molecule. The spontaneous breakdown of the dioxetanone releases the energy (about 50 kcal/mole) that is necessary to generate the excited state of the coelenteramide product, which is the singlet form of the monoanion. In vivo the product undergoes the process of nonradiative energy transfer to an accessory protein, a green fluorescent protein (GFP), which results in green bioluminescence. In vitro, in the absence of GFP, the product emits blue light.
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CAS REGISTRY NUMBER
COMMENTARY
346421-46-3
Gaussia luciferase
61869-41-8
-
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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
12-benzyl-8-hydroxy-2-(4-hydroxybenzyl)-5,11-dihydrobenzo[f]imidazo[1,2-a]quinoxalin-3(6H)-one + O2
oxidized 12-benzyl-8-hydroxy-2-(4-hydroxybenzyl)-5,11-dihydrobenzo[f]imidazo[1,2-a]quinoxalin-3(6H)-one + CO2 + hnu
-
-
-
-
?
2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
oxidized 2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one + CO2 + hn
-
-
-
-
?
2-benzyl-3-[(2-nitrophenyl)methoxy]-6-phenyl-8-(phenylsulfanyl)imidazo[1,2-a]pyrazine + O2
? + CO2
Renilla sp.
-
the substrate presents robust bioluminescent signals ex vivo and in living animals after UV irradiation at 365 nm
-
-
?
2-benzyl-3-[(4,5-dimethoxy-2-nitrophenyl)methoxy]-6-phenyl-8-(phenylsulfanyl)imidazo[1,2-a]pyrazine + O2
? + CO2
Renilla sp.
-
the substrate presents robust bioluminescent signals ex vivo and in living animals after UV irradiation at 365 nm
-
-
?
2-benzyl-3-[1-(2-nitrophenyl)ethoxy]-6-phenyl-8-(phenylsulfanyl)imidazo[1,2-a]pyrazine + O2
? + CO2
Renilla sp.
-
the substrate presents robust bioluminescent signals ex vivo and in living animals after UV irradiation at 365 nm
-
-
?
2-benzyl-8-benzyl-6-(2-fluorophenylethynyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
2-benzyl-8-benzyl-6-(3-fluorophenylethynyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
2-benzyl-8-benzyl-6-(3-hydroxyphenylethynyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
2-benzyl-8-benzyl-6-(3-methylphenylethynyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
2-benzyl-8-benzyl-6-(4-fluorophenylethynyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
2-benzyl-8-benzyl-6-(phenylethynyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
2-benzyl-8-benzyl-6-[(1-fluoroethyl)-1,2,3-triazol-4]imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
2-benzyl-8-benzyl-6-[(1-hydroxyethyl)-1,2,3-triazol-4]imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
2-benzyl-8-benzyl-6-[(1-hydroxypropyl)-1,2,3-triazol-4]imidazo[1,2-a]pyrazin-3(7H)-one + O2
? + CO2 + hv
-
-
-
?
8-benzyl-2-(4-fluorobenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
oxidized 8-benzyl-2-(4-fluorobenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one + CO2 + hnu
-
-
-
-
?
8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one + O2
oxidized 8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one + CO2 + hn
-
-
-
-
?
benzylluciferin methyl ether + O2
oxidized benzylluciferin methyl ether + CO2 + hv
-
-
-
-
?
bis-coelenterazine + O2
hnu + ?
-
assay at pH 7.6, about 100fold reduced relative luminescence
-
-
?
coelenterate-type luciferin + O2
oxidized coelenterate-type luciferin + CO2 + hv
-
-
-
-
?
coelenterazine-h + O2
coelenteramide h + CO2 + hv
substrate binding structure
-
-
?
coelenterazine-v + O2
coelenteramide-v + CO2 + hnu
increase of substrate coelenterazine stability by ligating it to Ca2+-triggered coelenterazine-binding protein, CBP, from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The apo-CBP binds coelenterazine-v very rapidly from Ca2+ free solution, similar to that as the native coelenterazine. At low concentrations, coelenterazine-v bound within CBP generates a brighter bioluminescence signal than would free coelenterazine, thereby increasing the assay sensitivity, overview
orange bioluminescence
-
?
cp-coelenterazine + O2
hnu + ?
-
assay at pH 7.6, about 100fold reduced relative luminescence
-
-
?
D-luciferin + O2 + ATP
oxidized D-luciferin + CO2 + H2O + AMP + diphosphate + hv
-
-
-
-
?
e-coelenterazine + O2
hnu + ?
-
assay at pH 7.6, about 100fold reduced relative luminescence
-
-
?
f-coelenterazine + O2
hnu + ?
-
assay at pH 7.6, about 100fold reduced relative luminescence
-
-
?
fcp-coelenterazine + O2
hnu + ?
-
assay at pH 7.6, about 100fold reduced relative luminescence
-
-
?
h-coelenterazine + O2
hnu + ?
-
assay at pH 7.6m, about 100fold reduced relative luminescence
-
-
?
hcp-coelenterazine + O2
hnu + ?
-
assay at pH 7.6, about 100fold reduced relative luminescence
-
-
?
MeO-coelenterazine + O2
hnu + ?
-
assay at pH 7.6, about 100fold reduced relative luminescence
-
-
?
n-coelenterazine + O2
hnu + ?
-
assay at pH 7.6, about 100fold reduced relative luminescence
-
-
?
3iso-coelenterazine + O2
?
-
relative activity to native coelenterazine: 78.2%
-
-
?
3iso-coelenterazine + O2
?
-
relative activity to native coelenterazine: 11.6%
-
-
?
3iso-coelenterazine + O2
?
-
relative activity to native coelenterazine: 34.9%
-
-
?
3me-coelenterazine + O2
?
-
relative activity to native coelenterazine: 80%
-
-
?
3me-coelenterazine + O2
?
-
relative activity to native coelenterazine: 11.8%
-
-
?
3me-coelenterazine + O2
?
-
relative activity to native coelenterazine: 45.5%
-
-
?
3meo-coelenterazine + O2
?
-
relative activity to native coelenterazine: 189%
-
-
?
3meo-coelenterazine + O2
?
-
relative activity to native coelenterazine: 5.5%
-
-
?
3meo-coelenterazine + O2
?
-
relative activity to native coelenterazine: 82.3%
-
-
?
alphameh-coelenterazine + O2
?
-
relative activity to native coelenterazine: 0.02%
-
-
?
alphameh-coelenterazine + O2
?
-
relative activity to native coelenterazine: 15.7%
-
-
?
alphameh-coelenterazine + O2
?
-
relative activity to native coelenterazine: 0.02%
-
-
?
alphameh-coelenterazine + O2
?
-
relative activity to native coelenterazine: 24.7%
-
-
?
cf3-coelenterazine + O2
?
-
relative activity to native coelenterazine: 49.5%
-
-
?
cf3-coelenterazine + O2
?
-
relative activity to native coelenterazine: 16.8%
-
-
?
cf3-coelenterazine + O2
?
-
relative activity to native coelenterazine: 5%
-
-
?
coelenterazine + O2
coelenteramide + CO2 + hnu
-
0.5 microg, assay at pH 7.6, high substrate specificity for coelenterazine
-
-
?
coelenterazine + O2
coelenteramide + CO2 + hnu
-
assay at pH 7.4, 25-30°C, 10 min
-
-
?
coelenterazine + O2
coelenteramide + CO2 + hnu
the enzyme catalyzes the oxidative decarboxylation of coelenterazine in the presence of O2, resulting in the formation of coelenteramide in its excited state and CO2 as the reaction product
green bioluminescence
-
?
coelenterazine + O2
coelenteramide + CO2 + hv
-
blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a complex of Ca2+-bound apoaequorin and coelenteramide, and shows luminescence activity like a luciferase. The oxidation of coelenterazine by BFP-aq in the luciferase reaction and the regeneration process to aequorin might involve the same catalytic site of BFP-aq
-
-
?
coelenterazine + O2
coelenteramide + CO2 + hv
-
clytin emits light on reacting with Ca2+. The Ca2+ sensitivity of recombinant clytin is lower than that of aequorin
-
-
?
coelenterazine + O2
coelenteramide + CO2 + hv
coelenterazine is bound by coelenterazine-binding protein. In combinantion with renilla luciferase, addition of only one Ca2+-ion is sufficient to release coelenterazine as a substrate. The combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone
-
-
?
coelenterazine + O2
coelenteramide + CO2 + hv
-
-
-
?
coelenterazine h + O2
excited coelenteramide h monoanion + CO2
-
-
-
?
coelenterazine h + O2
excited coelenteramide h monoanion + CO2
CAA01908.1
-
-
-
?
coelenterazine h + O2
excited coelenteramide h monoanion + CO2
an induced-fit mechanism is proposed where ligand-binding induces conformational changes of the active site. Insights regarding the controversial properties and the mechanism of the reaction catalysis of Renilla luciferase and its red-shifted light emittingvariant (Super RLuc 8)
-
-
?
et-coelenterazine + O2
?
-
relative activity to native coelenterazine: 21.1%
-
-
?
et-coelenterazine + O2
?
-
relative activity to native coelenterazine: 1.2%
-
-
?
et-coelenterazine + O2
?
-
relative activity to native coelenterazine: 78.3%
-
-
?
h-coelenterazine + O2
?
-
relative activity to native coelenterazine: 68.4%
-
-
?
h-coelenterazine + O2
?
-
relative activity to native coelenterazine: 12.4%
-
-
?
h-coelenterazine + O2
?
-
relative activity to native coelenterazine: 54.1%
-
-
?
i-coelenterazine + O2
?
-
relative activity to native coelenterazine: 32.3%
-
-
?
i-coelenterazine + O2
?
-
relative activity to native coelenterazine: 0.3%
-
-
?
i-coelenterazine + O2
?
-
relative activity to native coelenterazine: 5.1%
-
-
?
me-coelenterazine + O2
?
-
relative activity to native coelenterazine: 46.6%
-
-
?
me-coelenterazine + O2
?
-
relative activity to native coelenterazine: 5.2%
-
-
?
me-coelenterazine + O2
?
-
relative activity to native coelenterazine: 73.6%
-
-
?
meo-coelenterazine + O2
?
-
relative activity to native coelenterazine: 68.1%
-
-
?
meo-coelenterazine + O2
?
-
relative activity to native coelenterazine: 1.2%
-
-
?
meo-coelenterazine + O2
?
-
relative activity to native coelenterazine: 18.2%
-
-
?
Renilla luciferin + O2
oxidized Renilla luciferin + CO2 + hv
-
-
-
-
?
additional information
?
-
free coelenterazine and its analogues are unstable in neutral aqueous solution, undergoing a slow autooxidation over several hours
-
-
?
additional information
?
-
-
free coelenterazine and its analogues are unstable in neutral aqueous solution, undergoing a slow autooxidation over several hours
-
-
?
additional information
?
-
-
8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo-[1,2-a]pyrazin-3 (7H)-one is similar to, and as active as Renilla luciferin, the native luciferin has the benzyl group replaced by an unidentified group of approximately 200 Da
-
-
?
additional information
?
-
-
native 8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one and its derivatives are most promising substrates for use with Renilla luciferin reporter enzyme in cell culture and living animals
-
-
?
additional information
?
-
design and synthesis of bioluminescent coelenterazine derivatives (alkynes and triazoles) with imidazopyrazinone C-6 extended substitution as substrates for Renilla luciferase, evaluation, substrate specificity, overview
-
-
?
additional information
?
-
dynamic light scattering has been used for the detection of protein-protein interaction between the IgG antibody and modified enzyme FcUni-RLuc, to which an Fc-binding peptide is bound and separated by a five-amino acid linker from RLuc. Analysis of FcUni-RLuc and Herceptin interaction
-
-
?
additional information
?
-
establishment and evaluation of an indirect autophagy flux assay based on monitoring the degradation of an autophagosome-associated fusion protein Rluc-LC3 by luminescence detection. The Rluc-LC3 assay is useful for the identification of genes, miRNAs, and small molecules that regulate autophagy flux in mammalian cells. LC3 is a subfamily in the Atg8 family of ubiquitin-like proteins and is the only protein marker described to reliably bind the autophagosomal membrane and the phagophore Method evaluation and optimization, overview
-
-
?
additional information
?
-
substrate of Renilla luciferase, coelenterazine, is a heterocyclic imidazolo-pyrazinone, which is derivatized with (4-hydroxyphenyl)methyl (R2), 4-hydroxyphenyl (R6), and phenyl-methyl (R8) moieties
-
-
?
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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
coelenterazine + O2
coelenteramide + CO2 + hv
-
-
-
?
Renilla luciferin + O2
oxidized Renilla luciferin + CO2 + hv
-
-
-
-
?
coelenterazine h + O2
excited coelenteramide h monoanion + CO2
-
-
-
?
coelenterazine h + O2
excited coelenteramide h monoanion + CO2
CAA01908.1
-
-
-
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Mg2+
-
aequorin binds to Mg2+ under physiological conditions which modulates its light emission. The binding of Mg2+ to aequorin prevents the molecule from aggregating and stabilizes it in the monomeric form. Aequorin has two Mg2+ binding sites located within the EF-hands
Ca2+
-
after treatment with excess EDTA to remove Ca2+ from BFP-aq, the blue fluorescence shifts to a greenish fluorescence. This greenish fluorescent protein (GFP-aq) is identified as a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1
Ca2+
all three Ca2+-binding loops of photoproteins bind calcium ions
Ca2+
-
dependence of mutant and wild-type aequorin luminescence on Ca2+ concentration.Variations of luminescence kinetics, which depend on three EF hands endowed with different calcium affinities, critically determine the amplitude of aequorin responses to biological calcium signals
Ca2+
-
clytin emits light on reacting with Ca2+. The Ca2+ sensitivity of recombinant clytin is lower than that of aequorin
Ca2+
Renilla sp.
-
Ca2+-induced interaction between calmodulin and M13 leads to intermolecular complementation of split Renilla luciferase
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(2Z)-3-[[4-(dimethylamino)cyclohexa-1,5-dien-1-yl]amino]-1-phenylprop-2-en-1-one
-
-
(3Z)-3-[[4-(dimethylamino)phenyl]methylidene]-1,3-dihydro-2H-indol-2-one
-
common name SU4312
2-(4-ethoxyphenyl)-4-[(4-pyridin-2-ylpiperazin-1-yl)carbonyl]quinoline
-
-
2-(4-methylphenyl)-4-[(4-pyrimidin-2-ylpiperazin-1-yl)carbonyl]quinoline
-
-
2-hydroxy-N'-[(1E)-(2-hydroxyphenyl)methylidene]benzohydrazide
-
common name SCS
4-(1,4-dioxa-8-azaspiro[4.5]dec-8-ylcarbonyl)-2-(4-ethoxyphenyl)quinoline
-
-
4-amino-6-[(E)-[4'-[(Z)-(8-amino-1-hydroxy-5,7-disulfonato-6,7-dihydronaphthalen-2-yl)diazenyl]-3-methylbiphenyl-4-yl]diazenyl]-5-hydroxy-2,3-dihydronaphthalene-1,3-disulfonate
-
common name Evans Blue