Information on EC 1.13.12.6 - Cypridina-luciferin 2-monooxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.13.12.6
-
RECOMMENDED NAME
GeneOntology No.
Cypridina-luciferin 2-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Cypridina luciferin + O2 = oxidized Cypridina luciferin + CO2 + hnu
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
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redox reaction
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-
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reduction
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-
-
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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.
CAS REGISTRY NUMBER
COMMENTARY hide
61969-99-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
Cypridina sp.
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Manually annotated by BRENDA team
fish obtained from the gulf of mexico
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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Producibility and relative specific activity are apparently reduced in Cluc mutated in the phosphorylation sites, although the thermostability and secretion efficiency are not affected. Defects in the glycosylation modification are not related to secretion process and stability of the protein
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
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
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-
-
-
?
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
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-
-
?
Cypridina luciferin + O2
?
show the reaction diagram
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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
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Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
DL-Cypridina luciferin + O2
oxidized DL-Cypridina luciferin + CO2 + hv
show the reaction diagram
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-
-
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?
Vargula hilgendorfii luciferin + O2
oxidized Vargula hilgendorfii luciferin + CO2 + hv
show the reaction diagram
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native or recombinant substrate protein
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?
additional information
?
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Cypridina sp.
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evaluation of a method of measuring activity of Cypridina luciferase under conditions which minimize complicating with a relatively simple photoelectric light integrator, detailed overview
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
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
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Cypridina luciferin + O2
oxidized Cypridina luciferin + CO2 + hv
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
Cypridina sp.
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activates the enzyme by about 50% at higher pH values
Na+
Cypridina sp.
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activates the enzyme by about 50% at lower pH values
additional information
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evidence that luciferase requires a divalent metal ion, possibly calcium for activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
alpha,alpha'-dipyridyl
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slight inhibition
Cypndina gamma-globulin
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Cypridina luciferase is completely inhibited by a rabbit antibody to Cypridina luciferase
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Sodium azide
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slight inhibition
sodiumdodecylsulfate
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approx. 1 mM, complete inactivation
Urea
Cypridina sp.
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at 5.0 M urea, the half-time of the enzyme is 45 min, at 6.0 M urea 8 min, and at 7.5 M urea, the enzyme is inactivated within 1 min. rReversible, non-competitive inhibition of the luciferase activity occurs at concentrations up to 1.5 M
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
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
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pH 7.4
0.00028 - 0.0016
Cypridina luciferin
0.0005
Luciferin
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0.00045 - 0.00052
Vargula hilgendorfii luciferin
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additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0147 - 26.7
Cypridina luciferin
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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recombinant luciferase exhibits 14% activity of wild-type Cypridina luciferase
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
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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
6.8
Cypridina sp.
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assay at
7.4
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assay at
7.5
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26
Cypridina sp.
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assay at
37
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.9
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calculated
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10000
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x * 10000, equilibrium sedimentation
11500
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x * 11500, SDS-PAGE
13700
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x * 13700, possibly a hexamer, amino acid analysis
52000 - 57000
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determination of diffusion and sedimentation constant, gel filtration, sedimentation equilibrium
61415
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x * 61415, deduced from gene sequence
63000
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biotin-CLuc, SDS-PAGE
68000
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gel filtration, equilibrium sedimentation
200000
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SDS-PAGE, reduced antibody-avidin conjugate of the conjugated enzyme (FBP-IgG)
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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The biotinylated luciferase is stable when stored at 4C.
37
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53 h, 50% residual activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, distilled water
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-80C, lyophilized Cypridina luciferin is stable for more than 1 year and the enzyme in acidic ethanol solution for at least 1 month
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4C, PBS buffer containing 0.1 M potassium phosphate (pH 7.2) and 0.15 M NaCl, 2 months, without significant loss of activity
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Cypridina luciferase occasionally loses almost all of its activity when chromatographed through the 90-cm Sephadex column, irrespective of whether the column is newly poured or has been used for a period. The loss also occurs with both newly dissolved and old solutions of Cypridina luciferase
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purified native Apogon luciferase gradually loses activity on standing in the cold over a period of weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
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
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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
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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
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native enzyme by fractional precipitation with acetone and ammonium sulfate, and dialysis, followed by gel filtration
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native enzyme from photogenic organs by fractional precipitation with acetone and ammonium sulfate, and dialysis, followed by gel filtration
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Ni affinity column chromatography, SA beads column chromatography, monomeric avidin column chromatography, and TSK G300SW gel filtration
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recombinant secreted enzyme from Nicotiana benthamiana BY-2 cell medium by ultrafiltration, buffer exchange gel filtration, anion exchange chromatography, and dialysis
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Rat-1 fibroblasts
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expressed in Saccharomyces cerevisiae
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expression in Escherichia coli and mammalian cells
expression in NIH 3T3 cell
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expression in Saccharomyces cerevisiae
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expression of a chimeric protein G-luciferase enzyme in COS-1 and CHO cells
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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
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recombinant expression from vector p35SHSPG in Nicotiana benthamiana BY-2 cells, luciferase is efficiently secreted from cells with its native signal peptide, two potential N-glycosylation sites are modified with plant-type oligosaccharide chains. Recombinant expression of the enzyme in Arabidopsis thaliana roots in the cell wall of intact cells and the apoplast of plasmolyzed cells but not in the protoplasm. Cluc contains its own secretion signal peptide, which results in efficient secretion and folding of Cluc
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recombinant expression of wild-type and mutant enzymes in COS-1 cells and in Pichia pastoris strain GS115
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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
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N182D
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site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant enzyme behaves similar compared to the wild-type
N182D/N404D
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site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type
N182D/S406A
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site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type
N404D
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site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant enzyme behaves similar to the wild-type
S406A
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site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows increased protein accumulation compared to the wild-type
T184A
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site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows increased protein accumulation compared to the wild-type
T184A/N404D
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site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over 2.5fold increased protein accumulation compared to the wild-type
T184A/S406A
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site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
biotechnology
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use of enzyme as a potent secreted reporter
diagnostics
molecular biology