Bifunctional role of leucine 300 of firefly luciferase in structural rigidity.
Firefly luciferase is susceptible to thermal inactivation, thereby its intracellular half-life decreased. Previous reports indicated that L300R mutation (LRR mutant) in E354R/Arg356double mutant (ERR mutant) from Lampyris turkestanicus luciferase has increased its thermal stability and rigidity through induction of some ionic bonds with Asp 270 and 271. Disruption of the deduced ionic bonds in an ultra-rigid mutant of firefly luciferase did not reverse the flexibility of the protein. In this study, we investigated the effects of this residue to find the truth behind an extraordinary increase in thermal stability and rigidity of luciferase after replacement of leucine 300 by arginine based on previous reports. For this purpose, L300R, L300K and L300E mutations were performed to compare the effects of these mutations on the native firefly luciferase. In spite of increase of intrinsic fluorescence of the mutants a slight increase in thermostability and retention of kinetic properties was observed. Based on our results, we can conclude that L300R mutation in LRR mutant accompanying with alteration in a flexible loop (352-359) increased thermostability and rigidity of luciferase.
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; Afghanistan Specialists in Medicine Association (ASMA), Afghanistan.
Farzad
F
Yousefi
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Farangis
F
Ataei
Department of Biotechnology, Institute of Science, High Technology & Environmental Science, Graduate University of Advanced Technology, Kerman, Iran.
Mojtaba
M
Mortazavi
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: saman_h@modares.ac.ir.
Saman
S
Hosseinkhani
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013696:Temperature
D049409:Luciferases, Firefly; D007930:Leucine
Leucine 300; Luciferase; Mutation