Information on EC 1.13.12.13 - Oplophorus-luciferin 2-monooxygenase

for references in articles please use BRENDA:EC1.13.12.13
Word Map on EC 1.13.12.13
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.13.12.13
-
RECOMMENDED NAME
GeneOntology No.
Oplophorus-luciferin 2-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Oplophorus luciferin + O2 = oxidized Oplophorus luciferin + CO2 + hnu
show the reaction diagram
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
Oplophorus-luciferin:oxygen 2-oxidoreductase (decarboxylating)
The luciferase from the deep sea shrimp Oplophorus gracilirostris is a complex composed of more than one protein. The enzyme's specificity is quite broad, with both coelenterazine and bisdeoxycoelenterazine being good substrates.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
-
the enzyme is a member of the coelenterazine-utilizing luciferases such as Renilla luciferase and Gaussia luciferase
malfunction
-
recombinant mutant nanoKAZ lacks the amino-terminal signal peptide is expressed in the cytoplasm, translocated to the cell membrane, and released into the culture medium through an endoplasmic reticulum-Golgi-independent pathway
physiological function
-
the secreted luciferase of the deep-sea shrimp Oplophorus gracilirostris catalyzes the oxidation of coelenterazine to emit blue light (lambda_max = 460 nm)
additional information
-
the coelenterazine-binding site and the catalytic site for the luminescence reaction might be in a central cavity of the beta-barrel structure of the catalytic subunit KAZ
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-hydroxy-2-methylimidazol[1,2-a]pyridine + O2
oxidized 3-hydroxy-2-methylimidazol[1,2-a]pyridine + CO2 + hn
show the reaction diagram
-
-
-
-
?
3iso-coelenterazine + O2
oxidized 3iso-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 78.2%
-
-
?
3me-coelenterazine + O2
oxidized 3me-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 80%
-
-
?
3meo-coelenterazine + O2
oxidized 3meo-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 189%
-
-
?
6h-coelenterazine + O2
? + CO2 + hv
show the reaction diagram
Q9GV45 AND Q9GV46
-
-
-
?
6h-coelenterazine + O2
oxidized 6h-coelenterazine + CO2 + hnu
show the reaction diagram
-
luminescence intensity (Imax): 0.8
-
-
?
6h-f-coelenterazine + O2
? + CO2 + hv
show the reaction diagram
Q9GV45 AND Q9GV46
-
-
-
?
6h-f-coelenterazine + O2
oxidized 6h-f-coelenterazine + CO2 + hnu
show the reaction diagram
-
luminescence intensity (Imax): 10.1
-
-
?
alphameh-coelenterazine + O2
oxidized alphameh-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 15.7%
-
-
?
bis-coelenterazine + O2
? + CO2 + hv
show the reaction diagram
Q9GV45 AND Q9GV46
-
-
-
?
bis-coelenterazine + O2
oxidized bis-coelenterazine + CO2 + hnu
show the reaction diagram
-
luminescence intensity (Imax): 10.3
-
-
?
bisdeoxycoelenterazine + O2
oxidized bisdeoxycoelenterazine + CO2 + hn
show the reaction diagram
-
-
-
-
?
bisdeoxycoelenterazine + O2
oxidized bisdeoxycoelenterazine + CO2 + hnu
show the reaction diagram
-
native enzyme, 79%, catalytic subunit 19kOLase, 114% of the activity with coelenterazine, respectively
-
-
?
cf3-coelenterazine + O2
oxidized cf3-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 49.5%
-
-
?
coelenterazine + O2
coelenteramide + CO2 + hv
show the reaction diagram
coelenterazine + O2
oxidized coelenterazine + CO2 + hn
show the reaction diagram
coelenterazine + O2
oxidized coelenterazine + CO2 + hnu
show the reaction diagram
-
-
-
-
?
et-coelenterazine + O2
oxidized et-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 21.5%
-
-
?
f-coelenterazine + O2
? + CO2 + hv
show the reaction diagram
Q9GV45 AND Q9GV46
-
-
-
?
f-coelenterazine + O2
oxidized f-coelenterazine + CO2 + hnu
show the reaction diagram
fumirazine + O2
? + CO2 + hv
show the reaction diagram
Q9GV45 AND Q9GV46
-
-
-
?
furimazine + O2
?
show the reaction diagram
h-coelenterazine + O2
? + CO2 + hv
show the reaction diagram
Q9GV45 AND Q9GV46
-
-
-
?
h-coelenterazine + O2
oxidized h-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 68.4%
-
-
?
h-coelenterazine + O2
oxidized h-coelenterazine + CO2 + hnu
show the reaction diagram
i-coelenterazine + O2
oxidized i-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 32.3%
-
-
?
me-coelenterazine + O2
oxidized me-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 46.6%
-
-
?
meo-coelenterazine + O2
oxidized meo-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 68.1%
-
-
?
Oplophorus luciferin + O2
oxidized Oplophorus luciferin + CO2 + hv
show the reaction diagram
additional information
?
-
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
coelenterazine + O2
coelenteramide + CO2 + hv
show the reaction diagram
Oplophorus luciferin + O2
oxidized Oplophorus luciferin + CO2 + hv
show the reaction diagram
additional information
?
-
Q9GV45 AND Q9GV46
Oplophorus luciferase shows broad substrate specificities for various coelenterazine analogues, and the substrate specificity is distinct from other coelenterazine-type luciferases including Renilla and Gaussia luciferases and the Ca2+-binding photoprotein aequorin
-
-
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
-
0.001 mM, 98% inhibition
iodoacetamide
-
1 mM, catalytic subunit kOLase, 50% residual activity, native enzyme, 80% residual activity
N-ethylmaleimide
-
0.1 mM, catalytic subunit kOLase, 1% residual activity, native enzyme, 81% residual activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00004 - 0.0013
bis-deoxycoelenterazine
0.00014 - 0.0037
coelenterazine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
0.1% luminescence activity expressed in Escherichia coli cells using pCold-ZZ-KAZ vector (with IgG-binding domain of protein A), precipitate; 100% luminescence activity expressed in Escherichia coli cells using pCold-ZZ-KAZ vector (with IgG-binding domain of protein A), supernatant; 2.7% luminescence activity expressed in Escherichia coli cells using pCold-KAZ vector (without IgG-binding domain of protein A), supernatant; 4.7% luminescence activity expressed in Escherichia coli cells using pCold-KAZ vector (without IgG-binding domain of protein A), precipitate
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
-
Western blot, expression in CHO cells
106000
-
full-length enzyme
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterooligomer
Q9GV45 AND Q9GV46
x * 19000, catalytic subunit, + x * 35000, noncatalytic subunit, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified mutated 19 kDa protein nanoKAZ, X-ray diffraction structure determination and analysis at 1.71-2.3.A resolution and dialysis
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
enzyme retains activity in culture medium for more than 15 h at 37C
55
-
enzyme shows high physical stability, retaining activity with incubation up to 55C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
form inclusion bodies of recombinant Escherichia coli, solubilization with 6 M urea
-
recombinant His-tagged catalytic subunit mutant nanoKAZ from Escherichia coli strain BL21 by nickel affinity chromatography and dialysis, wild-typeKAZ is purified from inclusion bodies using 6 M urea-nickel chelate affinity column chromatography
-
recombinant His-tagged catalytic wild-type and mutant enzymes and catalytic subunits from Escherichia coli by nickel affinity chromatgraphy
-
using Ni-NTA chromatography
-
using nickel-chelate affinity column
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of a cold induced expression vector in Escherichia coli cells that consists of a histidine tag sequence for nickel chelate affinity purification, IgG-binding domain of protein A (ZZ-domain) and the multiple cloning sites, the role of ZZ-domain as a solubilizing partner at 15C is demonstrated by expressing the imidazopyrazinone-type luciferase of Oplophorus
expressed in Escherichia coli
-
expression of 19 kDa subunit 19kOLase in Escherichia coli
-
recombinant expression and secretion of 19 kDa catalytic subunit mutant nanoKAZ from CHO-K1 cells. Mutant nanoKAZ lacks the amino-terminal signal peptide, is expressed in the cytoplasm, translocated to the cell membrane, and released into the culture medium through an endoplasmic reticulum-Golgi-independent pathway. Recombinant expression of His-tagged wild-type and mutant KAZs in Escherichia coli. The secreted proteins form aggregates
-
recombinant expression and secretion, using the signal peptide sequence of Gaussia luciferase, of catalytic 19 kDa protein KAZ of Oplophorus luciferase and its mutants in Escherichia coli and recombinant expression of His-tagged wild-type and mutant enzymes and KAZs in CHO-K1 cells
-
recombinant expression of His-tagged 19 kDa catalytic subunit mutant nanoKAZ in Escherichia coli strain BL21. In bacterial cells, wild-type KAZ is mainly expressed as inclusion bodies
-
the catalytic domain of Oplophorus luciferase (19kOLase) is expressed in Escherichia coli cells using the cold-induced vectors of pCold-KAZ. Cold induction for Escherichia coli cells is carried out at 15C
-
the fused proteinof nanoKAZ with IgG-binding domain (ZZ domain) is expressed in the cytoplasm of Escherichia coli cells as a soluble form and purified. A secretory expression of nanoKAZ in CHO-K1 cells is performed using nanoKAZ fused with the signal peptide sequence of Gaussia luciferase
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A33N
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
A4E
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows wild-type bioluminesce intensity
A4E/Q11R/A33K/V44I/A54F/P115E/Q124K/Y138I
-
mutant is 29000fold brighter than mutant N166R. Western blot analysis shows that mutant A4E/Q11R/A33K/V44I/A54F/P115E/Q124K/Y138I is produced more efficiently than mutant N166R in cells. The increased expression is consistent with improved enzyme stability at 37C, where the half-life of activity retention is increased 65fold over that of mutant N166R
A4E/Q11R/A33K/V44I/A54F/P115E/Q124K/Y138I/Q18L/F54I/F68Y/L72Q/M75K/I90V/L27V/K33N/K43R/Y68D
-
mutant Nluc: Nluc paired with furimazine produces 2.5 millionfold brighter luminescence in mammalian cells relative to Oluc-19 with coelenterazine. The luminescence produced by Nluc decays with a half-life more than 2 h, significantly longer than for mutant A4E/Q11R/A33K/V44I/A54F/P115E/Q124K/Y138I. Nluc increased luminescence is gained mostly through improvements in protein stability, where Nluc shows markedly greater retention of activity in lysates following incubation at 37C
A4E/Q11R/Q18L/L27V/A33N/K43R/V44I/A54I/F68D/L72Q/M75K/I90V/P115E/Q124K/Y138I/N166R
-
construction of mutant nanoKAZ, a mutant with 16 amino acid substitutions of the catalytic subunit, by site-directed mutagenesis, the mutant nanoKAZ lacks the amino-terminal signal peptide, crystal structure determination and analysis, structure-function relationship in nanoKAZ, overview. The truncation of 10 amino acid residues at N- and C-terminal regions of nanoKAZ causes a complete loss of luminescence activity. Both the alpha1-helix and beta11-strand in the nanoKAZ molecule might serve to stabilize the molecule, which is essential in catalyzing the luminescence reaction
A54I
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
A54I/Y138I
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
C164A
-
mutation does not significantly affect catalytic activity of subunit kOLase
C164G
-
mutation does not significantly affect catalytic activity of subunit kOLase
C164S
-
mutation does not significantly affect catalytic activity of subunit kOLase
F68D
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows wild-type bioluminesce intensity
I90V
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
K43R
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows wild-type bioluminesce intensity
L27V
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows reduced bioluminesce intensity compared to the wild-type
L72Q
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
M75K
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
P115E
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
Q11R
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
Q124K
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
Q18L
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows wild-type bioluminesce intensity
V44I
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
V44I/A54I
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows the mutant shows increased bioluminesce intensity compared to the wild-type
V44I/A54I/Y138I
-
site-directed mutagenesis, mutation of the catalytic subunit, catalytic subunit mutant, that shows 7fold higher activity than 16-aa-mutant nanoKAZ using coelenterazine, but these substitutions does not stimulate protein secretion from mammalian cells, nanoKAZ possessing the signal peptide sequence of Gaussia luciferase for secretion expressed efficiently into the culture medium of CHO-K1 cells
V44I/Y138I
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows wild-type bioluminesce intensity
Y138I
-
site-directed mutagenesis, mutation of the catalytic subunit, the mutant shows increased bioluminesce intensity compared to the wild-type
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
wild-typeKAZ is purified from inclusion bodies using 6 M urea-nickel chelate affinity column chromatography
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
useful reporter protein in various assay systems including reporter assays and immunoassays