Information on EC 1.13.12.6 - Cypridina-luciferin 2-monooxygenase

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The expected taxonomic range for this enzyme is: Coelomata

EC NUMBER
COMMENTARY
1.13.12.6
-
RECOMMENDED NAME
GeneOntology No.
Cypridina-luciferin 2-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Cypridina luciferin + O2 = oxidized Cypridina luciferin + CO2 + hnu
show the reaction diagram
light, mechanism
-
Cypridina luciferin + O2 = oxidized Cypridina luciferin + CO2 + hnu
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
Cypridina-luciferin:oxygen 2-oxidoreductase (decarboxylating)
Cypridina is a bioluminescent crustacea. The luciferins (and presumably the luciferases, since they cross-react) of some luminous fish (e.g. Apogon, Parapriacanthus, Porichthys) are apparently similar. The enzyme may be assayed by measurement of light emission.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Cypridina luciferase
-
-
-
-
Cypridina luciferase
-
-
Cypridina luciferin 2-monooxygenase
-
-
-
-
Cypridina noctiluca luciferase
-
-
Cypridina-type luciferase
-
-
-
-
Fbp
-
biotinylated Cypridina luciferase conjugated to far-red fluorescent indocyanine derivative
FBP-IgG
-
conjugate of reduced monoclonal antibody to conjugated enyzme FBP
luciferase (Cypridina luciferin)
-
-
-
-
PGE2-luciferase
-
recombinant luciferase conjugated with N-hydroxysuccinimidyl ester of prostaglandin E2
Vargula luciferase
-
-
CAS REGISTRY NUMBER
COMMENTARY
61969-99-1
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Cypridina sp.
-
-
-
Manually annotated by BRENDA team
fish obtained from the gulf of mexico
-
-
Manually annotated by BRENDA team
also called Vargula hilgendorfii
-
-
Manually annotated by BRENDA team
formerly Cypridina hilgendorfii
SwissProt
Manually annotated by BRENDA team
formerly Cypridina hilgendorfii
-
-
Manually annotated by BRENDA team
sea firefly, marine ostracod crustacean
-
-
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
(S)-Cypridina luciferin I + O2
oxidized S-Cypridina luciferin I + CO2 + hv
show the reaction diagram
-
-
-
-
?
1-{3-[6-(1H-indol-3-yl)-2-(1-methylpropyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl]propyl}guanidine + O2
N-[3-(3-carbamimidamidopropyl)-5-(1H-indol-3-yl)-1,4-dihydropyrazin-2-yl]-2-methylbutanamide + CO2 + hn
show the reaction diagram
Q75R40
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
Cypridina sp.
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
P17554
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
bioluminescence
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
bioluminescence, maximum wavelength emission at 465 nm
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
conjugated enzyme exhibits bimodal bioluminescence spectrum at 460 nm and 675 nm, exhibits higher light intensity in mouse blood than unconjugated enzyme, far-red emission peak dominates in blood while the 460 nm peak dominates in buffers
-
-
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
very low bioluminescence rate with biluciferyl
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
Cypridina luciferin is [3-[3,7-dihydro-6-(1H-indol-3-yl)-2-[(S)-1-methyl-6-propyl]-3-oxoimidazo[1,2-a]pyrazin-8-yl]propyl]guanidine, equimolar complex of oxyluciferin with luciferase in an excited state is the light-emitter of Cypridina bioluminescence
-
?
Cypridina luciferin + O2
?
show the reaction diagram
-
fusion enzyme + 10 mM luciferin (dissolved in ethanol in 30 mM Tris-HCl (pH 7.6) with 10 mM EDTA) with ATP (250 mM), CoA (250 microM), and MgCl2 (5 mM) in 100 mM Tris-HCl, pH 7.8
Cypridina luciferase fusion enzyme expressed in Escherichia coli is soluble but does not exhibit bioluminescence
-
-
DL-Cypridina luciferin + O2
oxidized DL-Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
?
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
bioluminescence
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
bioluminescence, maximum wavelength emission at 465 nm
-
?
Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
-
-
conjugated enzyme exhibits bimodal bioluminescence spectrum at 460 nm and 675 nm, exhibits higher light intensity in mouse blood than unconjugated enzyme, far-red emission peak dominates in blood while the 460 nm peak dominates in buffers
-
-
Cypridina luciferin + O2
?
show the reaction diagram
-
fusion enzyme + 10 mM luciferin (dissolved in ethanol in 30 mM Tris-HCl (pH 7.6) with 10 mM EDTA) with ATP (250 mM), CoA (250 microM), and MgCl2 (5 mM) in 100 mM Tris-HCl, pH 7.8
Cypridina luciferase fusion enzyme expressed in Escherichia coli is soluble but does not exhibit bioluminescence
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
evidence that luciferase requires a divalent metal ion, possibly calcium for activity
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Sodium azide
-
slight inhibition
sodiumdodecylsulfate
-
approx. 1 mM, complete inactivation
alpha,alpha'-dipyridyl
-
slight inhibition
additional information
-
not inhibited by N-ethylmaleimide, N-methylmaleimide, iodoacetamide and p-chloromercuribenzoate
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.03
-
1-{3-[6-(1H-indol-3-yl)-2-(1-methylpropyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl]propyl}guanidine
Q75R40
pH 7.4
0.0016
-
Cypridina luciferin
-
35 nM recombinant prostaglandin E2-luciferase diluted with 0.1 M Tris buffer (pH 7.4) containing 0.05% bovine serum albumin, 1 nM monoclonal anti-prostaglandin E2 antibody, incubation for 18 h, 4C, in the dark, addition of 1 microM Cypridina luciferin in 0.1 M Tris-HCl buffer (pH 7.4) containing 0.3 M sodium ascorbate and 0.02 M Na2SO3 for bioluminescence reading
0.0005
-
Luciferin
-
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
26.7
-
Cypridina luciferin
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
-
recombinant luciferase exhibits 14% activity of wild-type Cypridina luciferase
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6.5
8
-
no difference in normalized bioluminescence spectra of conjugated enzyme in 100 mM sodium phosphate buffer (pH 6.5), 100 mM Tris-HCl buffer (pH 7.4) or 100 mM Tris-HCl buffer (pH 8) each containing 0.1 M NaCl
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4.9
-
Q75R40
calculated
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
52000
57000
-
determination of diffusion and sedimentation constant, gel filtration, sedimentation equilibrium
63000
-
-
biotin-CLuc, SDS-PAGE
68000
-
-
gel filtration, equilibrium sedimentation
200000
-
-
SDS-PAGE, reduced antibody-avidin conjugate of the conjugated enzyme (FBP-IgG)
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 10000, equilibrium sedimentation; x * 11500, SDS-PAGE; x * 13700, possibly a hexamer, amino acid analysis
?
Q75R40
x * 61415, deduced from gene sequence
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4
-
-
The biotinylated luciferase is stable when stored at 4C.
37
-
Q75R40
53 h, 50% residual activity
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-80C, lyophilized Cypridina luciferin is stable for more than 1 year and the enzyme in acidic ethanol solution for at least 1 month
-
4C, PBS buffer containing 0.1 M potassium phosphate (pH 7.2) and 0.15 M NaCl, 2 months, without significant loss of activity
-
-15C, distilled water
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
biotinylated enzyme is incubated with 1 microM sodium metaperiodate in 0.1 M sodium acetate buffer (pH 5.2), reaction stopped by glycerol addition, loading onto a size exclusion column, oxidized biotinylated enzyme eluted with the same buffer, pooling of bioluminescent fractions, concentration by ultrafree-0.5 centrifugal filter device, addition of 10 mM HiLyte Fluor 647 hydrazine in 0.1 M sodium acetate buffer (pH 5.2), after incubation loading onto a size-exclusion column, elution with 0.1 M potassium phosphate buffer (pH 7.2) containing 0.15 M NaCl. Treatment of human Delta-like protein (Dlk-1) antibody with 2-mercaptoethylamine (cleaving hinge-region disulfide bonds between heavy chains of antibody molecules) and conjugation of half antibodies to maleimide-activated avidin and purification on a size-exclusion column results in a 200 kDa conjugate
-
Escherichia coli cell culture with expression vector is cooled on ice-water bath, expression induced, incubation for 18 h at 15C, cell concentration by centrifugation, solution in 50 mM Tris-HCl (pH 7.6) and 10 mM EDTA, disruption by sonication, centrifugation, SDS-PAGE
-
loading of recombinant prostaglandin E2 luciferase onto size-exclusion column, elution with 0.1 M potassium phosphate buffer, pH 7.2, and 0.15 M NaCl, fractions with bioluminescent activities (Cypridina luciferin solution, 0.1 M Tris-HCl, pH 7.4, 0.3 M sodium ascorbate, 0.2 M Na2SO3) are combined and stored at -30C
-
Ni affinity column chromatography, SA beads column chromatography, monomeric avidin column chromatography, and TSK G300SW gel filtration
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expressed in Rat-1 fibroblasts
-
expressed in Saccharomyces cerevisiae
-
expression in NIH 3T3 cell
-
expression in Saccharomyces cerevisiae
-
expression of human Delta-like protein (Dlk-1) antigen in HEK-293 cells, immunization of mice (BALB/c) with HEK293-Dlk-1 cells or with the expression vector that encodes full-length Dlk-1 cDNA
-
Staphylococcus aureus ZZ-domain gene of protein A is amplified by PCR using pEZZ18 plasmid, the gene for Cypridina luciferase (obtained by PCR) is fused to the C-terminus of the ZZ-domain in the vector to give the expression vector pCold-ZZ-VL, the vector is then expressed in Escherichia coli BL21 grown in Luria-Bertani broth at 37C
-
expression in Escherichia coli and mammalian cells
P17554
expression of a chimeric protein G-luciferase enzyme in COS-1 and CHO cells
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
construction of a cold-induced expression vector (pCold-ZZ-VL vector) in Escherichia coli cells that consists of a histidine tag sequence for nickel chelate affinity purification, IgG-binding domain of Staphylococcuss aureus protein A (ZZ-domain) and multiple cloning sites, fusing Cypridina luciferase to the ZZ-domain results in expression by Escherichia coli of a soluble cytoplasm enzyme but it is not bioluminescent (in contrast to other luciferases fused to the ZZ-domain)
additional information
-
conjugation of prostaglandin E2 to luciferase by producing the N-hydroxysuccinimidyl ester of prostaglandin E2 by reaction of N-hydroxysuccinimde and 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride and conjugation of the ester to luciferase (ratio 18/1) in 0.1 M potassium phosphate buffer, pH 7.2, with 0.15 M NaCl for 13 h on ice
additional information
-
biotynilation of the enzyme by attachment of an Avi-tag of a 16-residue peptide to the C-terminus of the luciferase, conjugation to the indocyanine derivative HiLyte Fluor 647 hydrazine via the glycol-chains of the enzyme
additional information
-
establishment and validation of a yeast reporter assay, for detection of endocrine disruptors and analysis of protein-protein interactions by the yeast two-hybrid system, using a secretory luciferase, Cypridina noctiluca luciferase CLuc, as an alternative to the conventional beta-galactosidase, the CLuc reporter assay in yeast is more sensitive and convenient than the conventional assay, determination of the transcriptional activity of hundreds of yeast promoter fragments, overview
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
analysis
-
use of enzyme as a reporter in a screening system of Saccharomyces cerevisiae to improve secretion efficiency in yeast
analysis
-
dual reporter assay using one luciferase for monitoring gene expression and a second as an internal control, based on secreted luciferases from Cypridina noctiluca and Gaussia princeps that do not require lysis or special equipment. The assay can be carried out sequentially, the activities are high and can be measured in a small volume and a simple procedure. Development of a one-tube reporter assay for the two enzymes
biotechnology
Q75R40
use of enzyme as a potent secreted reporter
diagnostics
-
labeling of prostaglandin E2 with the bioluminescent enzyme Cypridina luciferase linked to monoclonal anti-prostaglandin E2 antibody as enyzme-linked immunosorbent assay (ELISA) system, detection of 7.8-500 pg/ml prostaglandin E2
diagnostics
-
Cypridina luciferase is used as bioluminescence reporter enzyme in yeast, bacterial, and mammalian cell-based assays, enzyme activity is measured with a luminometer by addition of native or synthetic luciferin, also known as Vargulin, 0.5 microM in 10 mM Tris-HCl, pH 7.4
diagnostics
-
far-red luminescence imaging technology to visualize tumor specific antigens on cell surfaces in vitro (human hepato-cellular carcinoma cell line Huh-7) and in living bodies (mice xenografted with human Delta-like protein-positive Huh-7 cells), based on a far-red fluorescent indocyanine derivative conjugated to biotinylated Cypridina luciferase linked to an anti-human Delta-like protein (Dlk-1) monoclonal antibody (embryonic antigens expressed on the surface of many cancer cells) via biotin-avidin interaction
molecular biology
-
construction of a cold-induced expression vector (pCold-ZZ-VL vector) in Escherichia coli cells that results in soluble bioluminescent fusion enzyme with binding ability to monoclonal antibodies, however, the Cypridina luciferase fusion enzyme is soluble but not bioluminescent (in contrast to other luciferases fused to the ZZ-domain of Staphylococcuss aureus protein A)
analysis
-
intramolecular bioluminescence resonance energy transfer system consisting of a fusion protein of the enzyme and enhanced yellow fluorescent protein for investigating peptide processing in living cells