1.14.14.3 A74F site-directed mutagenesis, the mutant shows reduced activity and increased Km compared to the wild-type enzyme 671985 1.14.14.3 A74G site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme 671985 1.14.14.3 A75G site-directed mutagenesis, activity similar to the wild-type enzyme 657939 1.14.14.3 A75G/C106V/V173A site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum 658060 1.14.14.3 A75G/C106V/V173C site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum 658060 1.14.14.3 A75G/C106V/V173S site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum 658060 1.14.14.3 A75G/C106V/V173T site-directed mutagenesis, alpha-subunit residues, reduced activity compared to the wild-type enzyme, further red shift of emission spectrum 658060 1.14.14.3 A81H site-directed mutagenesis, residue of the alpha-subunit, mutant shows 13% of wild-type activity 657927 1.14.14.3 alphaDELTA262-290beta four times higher affinity for FMN than wild type 348608 1.14.14.3 alphaF114A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site 671978 1.14.14.3 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 671978 1.14.14.3 alphaF114S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site 671978 1.14.14.3 alphaF114Y site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme 671978 1.14.14.3 alphaF117A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site 671978 1.14.14.3 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 671978 1.14.14.3 alphaF117S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site 671978 1.14.14.3 alphaF117Y site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme 671978 1.14.14.3 alphaF327A site-directed mutagenesis, mutant activity is similar to the wild-type enzyme 671978 1.14.14.3 alphaF46A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site 671978 1.14.14.3 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 671978 1.14.14.3 alphaF46S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site 671978 1.14.14.3 alphaF46Y site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme 671978 1.14.14.3 alphaF49A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site 671978 1.14.14.3 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 671978 1.14.14.3 alphaF49S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme due to reduced hydrophobicity of the active site 671978 1.14.14.3 alphaF49Y site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme 671978 1.14.14.3 alphaF6A site-directed mutagenesis, mutant activity is similar to the wild-type enzyme 671978 1.14.14.3 alphaH44A decreased bioluminescence 348600 1.14.14.3 alphaH44A rapid decay of the 4a-hydroperoxy-4a,5-dihydroFMN intermediate enzyme, HFOOH, in the mutant 676321 1.14.14.3 alphaR107A lower affinity for FMNH 348607 1.14.14.3 alphaR107E lower affinity for FMNH 348607 1.14.14.3 alphaR107S lower affinity for FMNH 348607 1.14.14.3 C106A site-directed mutagenesis, catalytic properties are similar to the wild-type enzyme, mutant shows 60% of wild-type quantum yield 658050 1.14.14.3 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 657939 1.14.14.3 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 657939 1.14.14.3 D232G random mutagenesis, 63% of wild-type luminescence activity 657855 1.14.14.3 D262A 90% reduced activity with octanal, 36% reduced activity with decanal, activity with dodecanal as the wild-type 704579 1.14.14.3 D265A activity with octanal as the wild-type, 81% reduced activity with decanal, complete loss of dodecanal activity 704579 1.14.14.3 D271A complete loss of octanal and decanal activity, 18% reduced activity with dodecanal 704579 1.14.14.3 E175G random mutagenesis, the single point mutation leads to increased decay rate of the enzyme, 0.9% of wild-type luminescence activity 657855 1.14.14.3 E175G/N199D random mutagenesis, 0.1% of wild-type luminescence activity 657855 1.14.14.3 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 671985 1.14.14.3 E328D site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme 671985 1.14.14.3 E328F site-directed mutagenesis, the mutant shows reduced activity and increased Km compared to the wild-type enzyme 671985 1.14.14.3 E328H site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme 671985 1.14.14.3 E328L site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme 671985 1.14.14.3 E328Q site-directed mutagenesis, the mutant shows highly reduced activity and increased Km compared to the wild-type enzyme 671985 1.14.14.3 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 657923 1.14.14.3 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 657923 1.14.14.3 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 657923 1.14.14.3 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 657923 1.14.14.3 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 657923 1.14.14.3 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 657923 1.14.14.3 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 657923 1.14.14.3 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 657923 1.14.14.3 G284P site-directed mutagenesis, residue of the alpha-subunit, 1-2% of the wild-type activity 657923 1.14.14.3 H285A 26% reduced activity with octanal, 74% reduced activity with decanal, complete loss of dodecanal activity 704579 1.14.14.3 H44A 1.5% of wild-type activity 764657 1.14.14.3 H44D 2.1% of wild-type activity 764657 1.14.14.3 H44N 2.2% of wild-type activity 764657 1.14.14.3 H45A 1.7% of wild-type activity 764657 1.14.14.3 H4A/H45A 1.95% of wild-type activity 764657 1.14.14.3 H81A site-directed mutagenesis, residue of the beta-subunit, mutant shows 59% of wild-type activity 657927 1.14.14.3 H81A/E89D site-directed mutagenesis, residues of the beta-subunit, mutant shows 13% of wild-type activity 657927 1.14.14.3 H82A site-directed mutagenesis, residue of the beta-subunit, mutant shows 22% of wild-type activity 657927 1.14.14.3 K202R random mutagenesis, 95% of wild-type luminescence activity 657855 1.14.14.3 K274A 89% reduced activity with octanal, 21% reduced activity with decanal, 81% reduced activity with dodecanal 704579 1.14.14.3 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 704579 1.14.14.3 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 704579 1.14.14.3 M190T random mutagenesis, 29% of wild-type luminescence activity 657855 1.14.14.3 additional information codon optimization of the luxCDE and frp genes, e.g. adaptation of the bacterial protein to mammalian temperature of 37°C, detailed overview 713371 1.14.14.3 additional information immobilization of the FMN reductase-luciferase complex 689388 1.14.14.3 additional information improvement of a tagging system, allowing real-time monitoring in vivo and in vitro, for luciferase by construction of a highly active, constitutive promoter resulting in a 100fold higher recombinant activity compared to native activity 671489 1.14.14.3 additional information Saccharomyces cerevisiae recombinantly expressing the Vibrio harveyi luciferase produces bright and stable luminescence, transformed yeast strains can grow on 0.5% v/v Z-9-tetradecenal, but die on 0.005% v/v decanal 657751 1.14.14.3 additional information substrate specificities of mutant enzymes and wild-type enzyme, overview, changes in the kinetics and emission spectrum on mutation of the chromophore-binding platform 658060 1.14.14.3 R291A 77% reduced activity with octanal, 58% reduced activity with decanal, 71% reduced activity with dodecanal 704579 1.14.14.3 T198S random mutagenesis, 84% of wild-type luminescence activity 657855 1.14.14.3 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 658060 1.14.14.3 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 658060 1.14.14.3 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 658060 1.14.14.3 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 658060 1.14.14.3 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 658060 1.14.14.3 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 658060 1.14.14.3 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 658060 1.14.14.3 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 658060 1.14.14.3 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 658060 1.14.14.3 W277A 11% reduced activity with octanal, 50% reduced activity with decanal and dodecanal 704579 1.14.14.3 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 702318 1.14.14.3 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 702318 1.14.14.3 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 702318 1.14.14.3 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 702318 1.14.14.3 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 702318 1.14.14.3 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 702318