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1.13.12.7: firefly luciferase

This is an abbreviated version!
For detailed information about firefly luciferase, go to the full flat file.

Word Map on EC 1.13.12.7

Reaction

D-firefly luciferin
+
O2
 +
ATP
=
firefly oxyluciferin
 +
CO2
 +
AMP
 +
diphosphate
 +
hnu

Synonyms

AL1, AL2, beetle luciferase, CBG99luc, CBRluc, FFL, firefly luciferase, firefly luciferin luciferase, fluc, LpLuc1, LpLuc2, Luc, Luc1, Luc1-type luciferase, Luc2, Luc2-type luciferase, luciferase, luciferase (firefly luciferin), luciferase FM, luciferin, Luciola italica luciferase, lucPpe, lucPpy, orange light-producing luciferase, oxygen 4-oxidoreductase, PC3-Luc, Photinus luciferin 4-monooxygenase (ATP-hydrolyzing), Photinus pyralis luciferase, PML, PpLase, Ppy, Ppy GR-TS, Ppy RE-TS, PpyWT, PsntWT

ECTree

     1 Oxidoreductases
         1.13 Acting on single donors with incorporation of molecular oxygen (oxygenases)
             1.13.12 With incorporation of one atom of oxygen (internal monooxygenases or internal mixed-function oxidases)
                1.13.12.7 firefly luciferase

Engineering

Engineering on EC 1.13.12.7 - firefly luciferase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E270A
Amydetes vivianii
the mutation drastically decreases the spectral pH-sensitivity, and extends the activity profile above pH 9.0. These mutations also decrease the sensitivity to metals such as zinc, mercury and cadmium. The mutation may unwind a turn of the alpha-helix-10, indirectly increasing the interaction of the pH-sensor and other residues at the bottom of the luciferin binding site, stabilizing the green light emitting conformation
E270G
Amydetes vivianii
the mutation drastically decreases the spectral pH-sensitivity, and extends the activity profile above pH 9.0. These mutations also decrease the sensitivity to metals such as zinc, mercury and cadmium. The mutation may unwind a turn of the alpha-helix-10, indirectly increasing the interaction of the pH-sensor and other residues at the bottom of the luciferin binding site, stabilizing the green light emitting conformation
F227Y
the mutation causes dramatic redshift and temporal shift
F260L
the mutation results in a much less pH-sensitive enzyme displaying only a 10 nm redshift and broadening of the spectra at pH 6
I226A
the mutation causes dramatic redshift and temporal shift
E353Q/ins356R
emission of red and green light at pH 7.8, 20% less activity
E354K
emission of red and green light at pH 7.8, 26% higher activity
E354K/ins356R
emission of red and green light at pH 7.8, 20% higher activity
E354R
emission of red and green light at pH 7.8, 33% higher activity
E354R/ins356R
emission of red light at pH 7.8 compared to green emitted light by wild-type luciferase, 53% higher activity compared to wild-type luciferase
H245N
red- and green-emitting luciferase, increase in thermostability, about 25% decrease in relative activity
H431Y
red- and green-emitting luciferase, about 85% decrease in relative acitivity
L300E
specific activity is increased and Km-value is increased about 1.7fold compared with the wild-type enzyme
L300K
relative stability of mutant luciferase increases compared with native luciferase, mutation causes no effects on bioluminescence spectrum
L300R
relative stability of mutant luciferase increases compared with native luciferase, mutation causes no effects on bioluminescence spectrum, specific activity is increased as compared to wild-type enzyme
S284T
red-emitting luciferase, about 75% decrease in relative activity
S286N
red bioluminescence
F16L
lower pH-dependence of bioluminescence spectra, about 40% decrease in specific activity
F16L/A40S
bioluminescence spectra independent from pH in range of pH 6.0-7.8, about 80% decrease in specific activity
H433Y
-
higher affinity to monomethyloxyluciferin and dimethyloxyluciferin compared to the wild type enzyme
S118C
increased thermostability, about 30% increase in specific activity
Y35H
bioluminescence spectra independent from pH in range of pH 6.0-7.8, about 40% decrease in specific activity
Y35N
bioluminescence spectra independent from pH in range of pH 6.0-7.8, about 30% decrease in specific activity
E356R/V368A
-
mutant enzyme with significantly improved thermostability, more than 90% of the activity remains after incubation for 1 h at 45°C. Unlike the wild-type enzyme the mutant enzyme shows no change in the emission maximum of 568 nm even at pH 6.3
F227Y
Macrolampis sp2
-
the mutation results in a unique time-dependent shift from red to yellow-green
G228A
Macrolampis sp2
-
the mutation results in a large redshift
Y227A
Macrolampis sp2
-
the substitution results in a weak and unstable red mutant
A296C/A326C
-
specific activity 676% (compared to wild-type 100%), Km (ATP) decreased compared to wild-type, Km (D-luciferin) decreased compared to wild-type, optimal temperature 35° (wild-type 25°C), optimal pH 8.5 (wild-type pH 8), activity remains 54% at 40°C for 5 min (compared to wild-type 0%), t1/2: 40 min at 35°C (compared to wild-type 5 min)
A348V
-
Km-value for D-luciferin is 8.9fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 2fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 6.5fold higher compared to the Km-value of the wild-type enzyme
AC571TG
-
nonsense mutation
D436A
3.4fold increased activity compared to the wild type enzyme
D436E
50% activity compared to the wild type enzyme
D436G
D436N
50% activity compared to the wild type enzyme
D436S
2.2fold increased activity compared to the wild type enzyme
D436V
2% activity compared to the wild type enzyme
D474K
-
D476N mutation does not have any significant effect
D476N
-
D474K mutation destabilizes the protein. Flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation is much more flexible than wild-type
D476P
site-directed mutagenesis, the mutant shows decreased thermostability compared to the wild-type
DELTA438-550
-
the purified N-terminal domain 1-437 has luminescence activity by itself, and binds to substrates ATP and luciferin with reduced affinity
E311Q
site-directed mutagenesis, the mutant shows 65% specific activity compared to the wild-type
E345K/A215L
-
half-life at 37°C is 7.36 min, compared to 3.06 min for the wild-type enzyme
E345K/A215L/I232A/T214A
-
half-life at 37°C is 75.1 min, compared to 3.06 min for the wild-type enzyme
E345K/A215L/I232A/T214A/F295L
-
half-life at 37°C is 82.1 min, compared to 3.06 min for the wild-type enzyme
E345K/I232A/T214A
-
half-life at 37°C is 15.5 min, compared to 3.06 min for the wild-type enzyme
E345K/I232A/T214A/F295L/S420T
-
half-life at 37°C is 72.4 min, compared to 3.06 min for the wild-type enzyme
E345K/T214A
-
half-life at 37°C is 8.5 min, compared to 3.06 min for the wild-type enzyme
F14R
-
improved pH tolerance and stability up to 45°C, no decrease in specific activity relative to the recombinant wild type enzyme
F14R/L35Q/V182K/I232K/F465R
-
improved pH tolerance and stability up to 45°C, no decrease in specific activity relative to the recombinant wild type enzyme
F247A
-
Km-value for D-luciferin is 15.3fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 17.9fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 21.9fold higher compared to the Km-value of the wild-type enzyme
F247L
-
Km-value for D-luciferin is 8.3fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is nearly identical to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 1.4fold higher compared to the Km-value of the wild-type enzyme
F247Y
-
Km-value for D-luciferin is 1.2fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 128fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 2fold lower compared to the Km-value of the wild-type enzyme
F250G
F250S
F250T
37% specific activity at pH 7.8 compared to the wild type enzyme
F465R
-
improved pH tolerance and stability up to 45°C, no decrease in specific activity relative to the recombinant wild type enzyme
G246A
G246A/F250G
33% specific activity at pH 7.8 compared to the wild type enzyme
G246A/F250S
42% specific activity at pH 7.8 compared to the wild type enzyme
G246A/F250T
40% specific activity at pH 7.8 compared to the wild type enzyme
G315A
-
Km-value for D-luciferin is 13.3fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 625fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 7.5fold higher compared to the Km-value of the wild-type enzyme
G316A
-
Km-value for D-luciferin is 1.2fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 2.6fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 1.5fold lower compared to the Km-value of the wild-type enzyme
G341A
-
Km-value for D-luciferin is 4fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 625fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 1.3fold higher compared to the Km-value of the wild-type enzyme
G446I
turnover number is 8.4fold lower compared to wild-type value, KM-value for D-luciferin is 1.5fold lower compared to wild-type value, KM-value for MgATP2- is 2.2fold lower compared to wild-type value, the bioluminescence emission maximum is 554 nm, compared to 558 nm for the wild-type value. The ratio of turnover-number to KM-value for D-luciferyl-O-adenosine monophosphate is 5.1fold lower compared to wild-type value
H245A
-
Km-value for D-luciferin is identical to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 3fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 1.5fold higher compared to the Km-value of the wild-type enzyme
H245D
-
longest rise time known among single point mutants
H461D
relative specific activity is 57.6% as compared to wild-type enzyme. Mutation decreases ATP binding affinity, reduces the melting temperature of protein by around 25°C and shifts its optimum temperature of activity to 10°C
H489D
relative specific activity is 112% as compared to wild-type enzyme. Mutation introduces a new salt bridge between the C-terminal and N-terminal domains and increases protein rigidity but only slightly improves its thermal stability
H489K
relative specific activity is 115% as compared to wild-type enzyme. Mutation increases protein rigidity but only slightly improves its thermal stability
H489M
relative specific activity is 103% as compared to wild-type enzyme
H489P
site-directed mutagenesis, the mutant shows improved thermostability while maintaining its catalytic efficiency compared to that of wild-type luciferase, the overall rigidity and local rigidity of H489Pmutant are greatly strengthened
I147A
30% activity compared to the wild type enzyme
I147E
4% activity compared to the wild type enzyme
I147F
1.2fold increased activity compared to the wild type enzyme
I147G
3% activity compared to the wild type enzyme
I147K
4% activity compared to the wild type enzyme
I147L
4.7fold increased activity compared to the wild type enzyme
I147M
3.4fold increased activity compared to the wild type enzyme
I147Q
70% activity compared to the wild type enzyme
I147R
1% activity compared to the wild type enzyme
I147S
7% activity compared to the wild type enzyme
I147V
1.3fold increased activity compared to the wild type enzyme
I232K
-
improved pH tolerance and stability up to 45°C, no decrease in specific activity relative to the recombinant wild type enzyme
I232R
-
specific activity 75% (compared to wild-type 100%), Km (ATP) increased compared to wild-type, Km (D-luciferin) increased compared to wild-type, optimal temperature 25° (wild-type 25°C), optimal pH 8.5 (wild-type pH 8), activity remains 4% at 40°C for 5 min (compared to wild-type 0%), t1/2: 20 min at 35°C (compared to wild-type 5 min)
I232R/A296C/A326C
-
specific activity 358% (compared to wild-type 100%), Km (ATP) increased compared to wild-type, Km (D-luciferin) increased compared to wild-type, optimal temperature 35° (wild-type 25°C), optimal pH 8 (wild-type pH 8), activity remains 28% at 40°C for 5 min (compared to wild-type 0%), t1/2: 10 min at 35°C (compared to wild-type 5 min)
I351A
-
Km-value for D-luciferin is 5.7fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 1.5fold higher compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 1.8fold higher compared to the Km-value of the wild-type enzyme
I423L
I423L/D436G/L530R
ins356E
green and red emitting light at pH 7.8, at pH 5.5 red-emitting luciferase, 97% increase in specific activity
ins356K
green and red emitting light at pH 7.8, at pH 5.5 red-emitting luciferase, 18% decrease in specific activity
ins356Q
at pH 5.5 red-emitting luciferase, increase in thermostability, 6% decrease in specific activity
ins356R
green and red emitting light at pH 7.8, at pH 5.5 red-emitting luciferase, 12% decrease in specific activtiy
K329I
point mutation does not affect the orientation of critical residues in bioluminescence color determination. Thermostability and Km value for luciferin are decreased as compared to wild type enzyme. Intrinsic fluorescence and far-UV CD intensity in K329I mutant is decreased. Realative activity as compared to wild-type enzyme is 32%
K443A
turnover number is 2655 fold lower compared to wild-type value, KM-value for D-luciferin is 6.5fold lower compared to wild-type value, KM-value for MgATP2- is 3.3fold lower compared to wild-type value, the bioluminescence emission maximum is identical to the wild-type value. The ratio of turnover-number to KM-value for D-luciferyl-O-adenosine monophosphate is 181fold lower compared to wild-type value
K443A/K529A
turnover number is 1063700fold lower compared to wild-type value, KM-value for D-luciferin is 4.5fold higher compared to wild-type value, KM-value for MgATP2- is 3.5fold higher compared to wild-type value, the bioluminescence emission maximum is 596 nm, compared to 558 nm for the wild-type value. The ratio of turnover-number to KM-value for D-luciferyl-O-adenosine monophosphate is 858fold lower compared to wild-type value
K445Q
turnover number is 1.4fold higher compared to wild-type value, KM-value for D-luciferin is 1.7fold lower compared to wild-type value, KM-value for MgATP2- is 2.3fold lower compared to wild-type value, the bioluminescence emission maximum is 556 nm, compared to 558 nm for the wild-type value. The ratio of turnover-number to KM-value for D-luciferyl-O-adenosine monophosphate is 1.7fold higher compared to wild-type value
K529A
L287I
-
orange light emitting mutant, mutation does not affect the structural integrity and/or folding of luciferase
L342A
-
Km-value for D-luciferin is 8.7fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 1.2fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 7.5fold higher compared to the Km-value of the wild-type enzyme
L35Q
-
improved pH tolerance and stability up to 45°C, no decrease in specific activity relative to the recombinant wild type enzyme
L530A
20% activity compared to the wild type enzyme
L530D
1% activity compared to the wild type enzyme
L530F
10% activity compared to the wild type enzyme
L530H
1.4fold increased activity compared to the wild type enzyme
L530I
shows wild type enzyme activity
L530K
3.2fold increased activity compared to the wild type enzyme
L530R
L530S
1% activity compared to the wild type enzyme
L530V
50% activity compared to the wild type enzyme
L530Y
2% activity compared to the wild type enzyme
N229T
site-directed mutagenesis, the mutant shows 63% specific activity compared to the wild-type
Q283R
-
red light emitting mutant, mutation does not affect the structural integrity and/or folding of luciferase
Q338P
-
about 50% decrease in specific activity compared to wild-type luciferase
Q448A
turnover number is 1.9fold lower compared to wild-type value, KM-value for D-luciferin is 2.5fold lower compared to wild-type value, KM-value for MgATP2- is 2.1fold higher compared to wild-type value, the bioluminescence emission maximum is 557 nm, compared to 558 nm for the wild-type value. The ratio of turnover-number to KM-value for D-luciferyl-O-adenosine monophosphate is 1.1fold lower compared to wild-type value
R213E
-
same activity like wild-type luciferase
R213K/T214N
site-directed mutagenesis, residues K213 and/or N214 are largely responsible for the 1.55fold increase in specific activity of the variant
R213M
R218A
-
Km-value for D-luciferin is 20fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 31.3fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 38.8fold higher compared to the Km-value of the wild-type enzyme
R218Q
site-directed mutagenesis, the mutant shows 125% specific activity compared to the wild-type
R330Q
point mutation does not affect the orientation of critical residues in bioluminescence color determination. Thermostability and Km value for luciferin are decreased as compared to wild type enzyme. Increase in tryptophan fluorescence intensity and secondary structure content for R330Q as compared with wild type. Realative activity as compared to wild-type enzyme is 23%
R337Q
S184T
-
red-emitting luciferase with a bioluminescence maximum of 615 nm
S284G
-
red light emitting mutant, mutation does not affect the structural integrity and/or folding of luciferase
S284T
S293P
-
orange light emitting mutant, mutation does not affect the structural integrity and/or folding of luciferase
S307P/H489P
site-directed mutagenesis, the mutation is randomly chosen outside the flexible regions as a control. The mutant has decreased kinetic stability and enhanced thermodynamic stability compared to the wild-type
S347A
-
Km-value for D-luciferin is 11.3fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 4.2fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 2.2fold higher compared to the Km-value of the wild-type enzyme
S440P/S456Y
-
mutant shows improved specificity and reactivity for ATP compared to wild-type
S440R/S456A
-
mutant shows improved specificity and reactivity for ATP compared to wild-type
S440R/S456V
-
mutant shows improved specificity for ATP compared to wild-type
T214A/A215L/I232A/F295L/E345K
the mutant enzyme displays high thermostability, retaining about 60% activity after 120 min at 45°C. Although the mutant shows higher maximum activity at high D-luciferin concentrations, its activity at low D-luciferin concentrations (below 0.004 mM) lags behind that of the mutantI423L/D436G/L530R
T214A/A215L/I232A/F295L/E345K/I423L/D436G/L530R
the mutant enzyme exhibits both improved thermostability and brighter luminescence at low luciferin concentrations, it may be useful for reporter gene applications
T251A
-
Km-value for D-luciferin is 20.7fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 2.9fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 12.5fold higher compared to the Km-value of the wild-type enzyme
T343A
-
Km-value for D-luciferin is 6.6fold higher compared to the Km-value of the wild-type enzyme, the turnover-number for D-luciferin is 125fold lower compared to the turnover-number of the wild-type enzyme, the Km-value for MgATP2- is 5.4fold higher compared to the Km-value of the wild-type enzyme
V182K
-
improved pH tolerance and stability up to 45°C, no decrease in specific activity relative to the recombinant wild type enzyme
V241I
136% specific activity at pH 7.8 compared to the wild type enzyme
V241I/F250G
13% specific activity at pH 7.8 compared to the wild type enzyme
V241I/F250S
32% specific activity at pH 7.8 compared to the wild type enzyme
V241I/F250T
64% specific activity at pH 7.8 compared to the wild type enzyme
V241I/G246A/F250S
73% specific activity at pH 7.8 compared to the wild type enzyme
V241I/G246A/F250T
83% specific activity at pH 7.8 compared to the wild type enzyme
Y255F
site-directed mutagenesis, the mutant shows 71% specific activity compared to the wild-type
F255Y
site-directed mutagenesis, the single change Y255F is necessary and sufficient for a 13 nm redshift in bioluminescence
Y227A
mutant shows enzymatic behaviour similar to the wild type enzyme
G228A
the mutation results in a large redshift
Y227A
the mutation causes dramatic red- and time-dependent shifts
L260F
-
mutation results in a 20 nm redshift without affecting pH-insensitivity
V227A
-
mutant displays a small redshift
V227F
-
mutation results in a 60 nm redshift
V227I
-
mutant displays a small redshift
V227S
-
mutant displays a small redshift
A243G
-
bioluminescence emission maximum with luciferyl-O-adenosine monophosphate is 599 nm compared to 549 nm for the wild-type enzyme, bioluminescence emission maxima with 5,5-dimethyl-luciferyl-O-adenosine monophosphate are 610 nm and 557 nm compared to 624 nm for the wild-type enzyme
S247F
-
bioluminescence emission maximum with luciferyl-O-adenosine monophosphate is 597 nm compared to 549 nm for the wild-type enzyme, bioluminescence emission maximum with 5,5-dimethyl-luciferyl-O-adenosine monophosphate is 612 nm compared to 624 nm for the wild-type enzyme
additional information