title: The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase.
abstract: The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.
authors.affiliation: Department of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran 14115-175, Iran.
authors.forename: Narges Kh
authors.initials: NKh
authors.lastname: Tafreshi
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
authors.forename: Majid
authors.initials: M
authors.lastname: Sadeghizadeh
authors.forename: Mehdi
authors.initials: M
authors.lastname: Sadeghi
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
authors.forename: Hossein
authors.initials: H
authors.lastname: Naderi-Manesh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D048888:Fireflies; D007700:Kinetics; D049409:Luciferases, Firefly / Q000235:genetics* / Q000378:metabolism*; D049449:Luminescence; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D001120:Arginine; D049409:Luciferases, Firefly