title: Design of thermostable luciferases through arginine saturation in solvent-exposed loops.
abstract: In most bioluminescence systems the oxidation of luciferin and production of light is catalyzed by luciferases. Protein engineering studies have shown that thermostable proteins from thermophilic organisms have a higher frequency of Arg, especially in exposed states. To further clarify the arginine saturation effect on thermostability of firefly luciferase, some of hydrophobic solvent-exposed residues in Lampyris turkestanicus luciferase are changed to arginine. All of these residues are located at the surface loops of L.turkestanicus luciferase. Starting with a luciferase mutant (E³⁵⁴Q/Arg³⁵⁶), single mutation (-Q³⁵R, -I¹⁸²R, -I²³²R and -L(300)R), double mutation (-Q³⁵R/I²³²R) and triple mutation (-Q³⁵R/I²³²R/I¹⁸²R) are added. Bioluminescence emission spectra indicate that substitution of Arg by these residues, do not effect on the maximum wavelength of emission spectrum. It should be noted, introduction of double mutation (-Q³⁵R/I²³²R) and triple mutation (-Q³⁵R/I²³²R/I¹⁸²R) were kept specific activity of firefly luciferase. By addition of positively charged residue, some specific mutations (-I²³²R, -Q³⁵R/I²³²R and -Q³⁵R/I²³²R/I¹⁸²R) showed that optimum temperature of activity was increased to 40°C which are 12 and 15°C higher than E³⁵⁴Q/Arg³⁵⁶ and wild-type luciferases, respectively. Also, after 40 min incubation of enzymes at 40°C, the relative remaining activity of wild type was only 5%, whereas for -I²³²R, -Q³⁵R/I²³²R and -Q³⁵R/I²³²R/I¹⁸²R was 60, 80 and 80% of original activity, respectively.
authors: Department of Biochemistry, Faculty of Biological Sciences,  Tarbiat Modares University, Tehran,  Iran.
authors: Mojtaba
authors: M
authors: Mortazavi
authors: Saman
authors: S
authors: Hosseinkhani
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics*; D002942:Circular Dichroism; D004795:Enzyme Stability; D048888:Fireflies; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics; D049449:Luminescence; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D001120:Arginine; D049409:Luciferases, Firefly