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1.14.14.3: bacterial luciferase

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

Word Map on EC 1.14.14.3

Reaction

a long-chain aldehyde
+
FMNH2
+
O2
=
a long-chain fatty acid
+
FMN
+
H2O
+
hnu

Synonyms

4a-hydroperoxy-4a,5-dihydroFMN intermediate luciferase, aldehyde monooxygenase, alkanal monooxygenase (FMN), bacterial luciferase, COB, Gluc luciferase, HFOOH, luciferase, Lux, LuxA, LuxAB, LuxB, LuxCDABE, LuxF, Vibrio fischeri luciferase, Vibrio harveyi luciferase

ECTree

     1 Oxidoreductases
         1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
             1.14.14 With reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen into the other donor
                1.14.14.3 bacterial luciferase

Engineering

Engineering on EC 1.14.14.3 - bacterial luciferase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D232G
-
random mutagenesis, 63% of wild-type luminescence activity
E175G
-
random mutagenesis, the single point mutation leads to increased decay rate of the enzyme, 0.9% of wild-type luminescence activity
E175G/N199D
-
random mutagenesis, 0.1% of wild-type luminescence activity
K202R
-
random mutagenesis, 95% of wild-type luminescence activity
M190T
-
random mutagenesis, 29% of wild-type luminescence activity
T198S
-
random mutagenesis, 84% of wild-type luminescence activity
A74F
-
site-directed mutagenesis, the mutant shows reduced activity and increased Km compared to the wild-type enzyme
A74G
-
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
A75G
-
site-directed mutagenesis, activity similar to the wild-type enzyme
A75G/C106V/V173A
-
site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum
A75G/C106V/V173C
-
site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum
A75G/C106V/V173S
-
site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum
A75G/C106V/V173T
-
site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum
A81H
-
site-directed mutagenesis, residue of the alpha-subunit, mutant shows 13% of wild-type activity
alphaDELTA262-290beta
-
four times higher affinity for FMN than wild type
alphaF114A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF114D
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF114S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF114Y
-
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
alphaF117A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF117D
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF117S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF117Y
-
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
alphaF327A
-
site-directed mutagenesis, mutant activity is similar to the wild-type enzyme
alphaF46A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF46D
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF46S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF46Y
-
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
alphaF49A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF49D
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF49S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site
alphaF49Y
-
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
alphaF6A
-
site-directed mutagenesis, mutant activity is similar to the wild-type enzyme
alphaH44A
alphaR107A
-
lower affinity for FMNH
alphaR107E
-
lower affinity for FMNH
alphaR107S
-
lower affinity for FMNH
C106A
-
site-directed mutagenesis, catalytic properties are similar to the wild-type enzyme, mutant shows 60% of wild-type quantum yield
C106V
-
site-directed mutagenesis, highly reduced ability to stabilize the reaction intermediate due to interaction between Val106 and Ala75 side chains, and therefore highly reduced activity and increased thermal lability compared to the wild-type enzyme
C106V/A75G
-
site-directed mutagenesis, mutation of Ala75 restores about 90% of the activity abolished by mutation of Cys106, shift in the light emission spectrum to that of Photobacterium phosphoreum possessing Val and Gly at positions 106 and 75, respectively
D262A
-
90% reduced activity with octanal, 36% reduced activity with decanal, activity with dodecanal as the wild-type
D265A
-
activity with octanal as the wild-type, 81% reduced activity with decanal, complete loss of dodecanal activity
D271A
-
complete loss of octanal and decanal activity, 18% reduced activity with dodecanal
E328A
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme, the activity is rescued by addition of sodium acetate, but not by phosphate, at pH 6.0-8.0 with increasing activity at lower pH
E328D
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme
E328F
-
site-directed mutagenesis, the mutant shows reduced activity and increased Km compared to the wild-type enzyme
E328H
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme
E328L
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme
E328Q
-
site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme
F261A
-
site-directed mutagenesis, residue of the alpha-subunit, 0.19% of the wild-type activity, the bulky and hydrophobic nature of the alphaF261 residue is critical for activity
F261D
-
site-directed mutagenesis, residue of the alpha-subunit, 0.004% of the wild-type activity, the bulky and hydrophobic nature of the alphaF261 residue is critical for activity
F261S
-
site-directed mutagenesis, residue of the alpha-subunit, 0.13% of the wild-type activity, the bulky and hydrophobic nature of the alphaF261 residue is critical for activity
F261Y
-
site-directed mutagenesis, residue of the alpha-subunit, 2-3% of the wild-type activity, the bulky and hydrophobic nature of the alphaF261 residue is critical for activity
G275A
-
site-directed mutagenesis, residue of the alpha-subunit, 27% of the wild-type activity, the torsional flexibility of the alphaG275 residue is critical for activity
G275F
-
site-directed mutagenesis, residue of the alpha-subunit, 6-7% of the wild-type activity, the torsional flexibility of the alphaG275 residue is critical for activity
G275I
-
site-directed mutagenesis, residue of the alpha-subunit, 15% of the wild-type activity, the torsional flexibility of the alphaG275 residue is critical for activity
G275P
-
site-directed mutagenesis, residue of the alpha-subunit, 0.04% of the wild-type activity, the torsional flexibility of the alphaG275 residue is critical for activity
G284P
-
site-directed mutagenesis, residue of the alpha-subunit, 1-2% of the wild-type activity
H285A
-
26% reduced activity with octanal, 74% reduced activity with decanal, complete loss of dodecanal activity
H44A
1.5% of wild-type activity
H44D
2.1% of wild-type activity
H44N
2.2% of wild-type activity
H45A
1.7% of wild-type activity
H4A/H45A
1.95% of wild-type activity
H81A
-
site-directed mutagenesis, residue of the beta-subunit, mutant shows 59% of wild-type activity
H81A/E89D
-
site-directed mutagenesis, residues of the beta-subunit, mutant shows 13% of wild-type activity
H82A
-
site-directed mutagenesis, residue of the beta-subunit, mutant shows 22% of wild-type activity
K274A
-
89% reduced activity with octanal, 21% reduced activity with decanal, 81% reduced activity with dodecanal
K283A
-
complete loss of octanal and decanal activity, 96% reduced activity with dodecanal, does not significantly impede binding of decanal, results in destabilization of intermediate II, results in a loss in quantum yield comparable with that of the loop deletion mutant, binds reduced flavin more weakly
K286A
-
92% reduced activity with octanal, complete loss of decanal activity, 87% reduced activity with dodecanal, does not significantly impede binding of decanal, increase in exposure of reaction intermediates to a dynamic quencher, results in a loss in quantum yield comparable with that of the loop deletion mutant, binds reduced flavin more weakly
R291A
-
77% reduced activity with octanal, 58% reduced activity with decanal, 71% reduced activity with dodecanal
V173A
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173C
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173F
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173H
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173I
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173L
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173N
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173S
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
V173T
-
site-directed mutagenesis, alpha-subunit residue, reduced activity and decreased stability of the C4a-hydroperoxyflavin intermediate compared to the wild-type enzyme, red shift of emission spectrum
W277A
-
11% reduced activity with octanal, 50% reduced activity with decanal and dodecanal
Y151A
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151D
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151K
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151R
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151T
binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
Y151W
least active mutant, binds reduced flavin with wild-type affinity, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield
additional information