Information on EC 1.13.12.13 - Oplophorus-luciferin 2-monooxygenase

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The expected taxonomic range for this enzyme is: Coelomata

EC NUMBER
COMMENTARY
1.13.12.13
-
RECOMMENDED NAME
GeneOntology No.
Oplophorus-luciferin 2-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY
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 gracilorostris is a complex composed of more than one protein. The enzyme's specificity is quite broad, with both coelenterazine and bisdeoxycoelenterazine being good substrates.
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
imidazopyrazinone-type luciferase
Q9GV45
-
nanoKAZ
-
mutated catalytic component of Oplophorus luciferase
Oplophorus luciferase
-
-
-
-
Oplophorus luciferase
-
-
Oplophorus luciferase
Q9GV45
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
deep-sea shrimp
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
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
oxidized 6h-coelenterazine + CO2 + hnu
show the reaction diagram
-
luminescence intensity (Imax): 0.8
-
-
?
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
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
oxidized coelenterazine + CO2 + hn
show the reaction diagram
-
-
-
-
?
coelenterazine + O2
oxidized coelenterazine + CO2 + hn
show the reaction diagram
-
-
-
-
?
coelenterazine + O2
oxidized coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intensity (Imax): 1.0
-
-
?
coelenterazine + O2
oxidized coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 100%
-
-
?
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
oxidized f-coelenterazine + CO2 + hnu
show the reaction diagram
-
native enzyme, 26%, catalytic subunit 19kOLase, 80% of the activity with coelenterazine, respectively
-
-
?
f-coelenterazine + O2
oxidized f-coelenterazine + CO2 + hnu
show the reaction diagram
-
luminescence intensity (Imax): 19.5
-
-
?
furimazine + O2
?
show the reaction diagram
-
-
-
-
?
furimazine + O2
?
show the reaction diagram
-
luminescence intensity (Imax): 10.1
-
-
?
h-coelenterazine + O2
oxidized h-coelenterazine + CO2 + hnu
show the reaction diagram
-
native enzyme, 97%, catalytic subunit 19kOLase, 58% of the activity with coelenterazine, respcetively
-
-
?
h-coelenterazine + O2
oxidized h-coelenterazine + CO2 + hnu
show the reaction diagram
-
luminescence intensity (Imax): 17
-
-
?
h-coelenterazine + O2
oxidized h-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 68.4%
-
-
?
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%
-
-
?
Oplophorus luciferin + O2
oxidized Oplophorus luciferin + CO2 + hv
show the reaction diagram
-
-
-
-
?
meo-coelenterazine + O2
oxidized meo-coelenterazine + CO2 + hn
show the reaction diagram
-
luminescence intesity (Imax): 68.1%
-
-
?
additional information
?
-
-
luminescence intensity of the catalytic subunit 19kOLase alone is seven times lower for coelenterazine and three times lower for biscoelenterazine than that of native Oplophorus luciferase, respectively
-
-
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
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
LITERATURE
IMAGE
0.00004
bis-deoxycoelenterazine
-
pH 7.6, native enzymee
0.0013
bis-deoxycoelenterazine
-
pH 7.6, catalytic subunit 19kOLase
0.00014
coelenterazine
-
pH 7.6, native enzyme
0.0037
coelenterazine
-
pH 7.6, catalytic subunit 19kOLase
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
Q9GV45
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
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20000
-
Western blot, expression in CHO cells
726882
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
enzyme consists of 19 and 35 kDa proteins. Luminescence intensity of the catalytic subunit 19kOLase alone is seven times lower for coelenterazine and three times lower for biscoelenterazine than that of native Oplophorus luciferase, respectively
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
-
enzyme retains activity in culture medium for more than 15 h at 37C
727290
55
-
enzyme shows high physical stability, retaining activity with incubation up to 55C
727290
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
form inclusion bodies of recombinant Escherichia coli, solubilization with 6 M urea
-
using Ni-NTA chromatography
-
using nickel-chelate affinity column
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
-
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
Q9GV45
expression of 19 kDa subunit 19kOLase in Escherichia coli
-
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
LITERATURE
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
N166R
-
mutant shows 50% increased stability at 37C and 3fold higher luminescence intensity
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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
molecular biology
Q9GV45
useful reporter protein in various assay systems including reporter assays and immunoassays