International Journal of Biological Macromolecules 101 (2017) 67–74 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Bifunctional role of leucine 300 of ﬁreﬂy luciferase in structural rigidity Farzad Youseﬁ a,b , Farangis Ataei a , Mojtaba Mortazavi c , Saman Hosseinkhani a,∗ a Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran b Afghanistan Specialists in Medicine Association (ASMA), Afghanistan c Department of Biotechnology, Institute of Science, High Technology & Environmental Science, Graduate University of Advanced Technology, Kerman, Iran a r t i c l e i n f o Article history: Received 17 October 2016 Received in revised form 25 February 2017 Accepted 14 March 2017 Available online 18 March 2017 Keywords: Luciferase Leucine 300 Mutation a b s t r a c t Fireﬂy luciferase is susceptible to thermal inactivation, thereby its intracellular half-life decreased. Pre- vious reports indicated that L 300 R mutation (LRR mutant) in E 354 R/Arg 356 double 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, L 300 R, L 300 K and L 300 E 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 L 300 R muta- tion in LRR mutant accompanying with alteration in a ﬂexible loop (352–359) increased thermostability and rigidity of luciferase. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Fireﬂy luciferase is a peroxisomal enzyme that converts a cyclic substrate luciferin to an excited state oxyluciferin in the presence of ATP, Mg +2 and O 2 [1,2] . When excited state oxyluciferin molecules return to ground state, light is produced with high quantum yield [3,4]. Luciferase from Photinus pyralis produces light at pH ≈ 7.6 in vitro, in the range of green-yellow ( max 557 nm) and red ( max 620 nm) at pH ≈ 5.4 [5–7]. Naturally, most ﬁreﬂy species emit light in yellow-green and few in red. Fireﬂy luciferase-based assay is a highly sensitive technique and has wide applications such as ATP assay [8–10], in vivo imaging [8], gene reporters [11], Pyrosequenc- ing [12] and luciferase-based split biosensors [13]. In order to exploit luciferase in different applications following strategies usually applied including: optimization and stabiliza- tion of kinetic properties through rational and random mutations [14,15], enzyme inhibitors removing by addition of detergents [16], Coenzyme A (CoA) and other thiol compounds [17,18], pyruvate orthophosphate dikinase (PPDK) [19], and luciferin regenerating ∗ Corresponding author. E-mail address: saman h@modares.ac.ir (S. Hosseinkhani). enzyme (LRE) to recycle adenosine monophosphate (AMP) and oxy- luciferin [20–23] as luciferase reaction products, respectively. Luciferase is a mesophilic enzyme which exhibit low temper- ature stability in vivo and in vitro. Thus, many studies have been performed to overcome this problem. For this purpose, luciferases sequences and structures have been compared with those of many thermophile proteins and based on the data obtained, different experiments have been designed to increase luciferase thermosta- bility [14,24–27]. According to the aforementioned studies in thermostable proteins, guanidinium group of arginine residue situ- ated at the protein surface loops, participate in ionic and hydrogen interactions which in turn lead to unexpected increase in ther- mal stability [14,27]. For example, in acetylcholine esterase 14 hydrophobic residues situated at surface loops and their replace- ment by arginine resulted in increase in thermal stability [28]. Mutation in the most ﬂexible region of ﬁreﬂy luciferase (D 474 K and D 476 N) shows that D 474 K mutation became much more ﬂexible than wild type although D 476 N didn’t have any signiﬁcant differ- ence [29]. Moreover, H 489 P mutation within most ﬂexible regions of luciferase improved its thermostability while maintaining its cat- alytic efﬁciency compared to that of wild type luciferase. Rigidity of H 489 P mutant is greatly strengthened. D 476 P mutation decreased its thermostability while S 307 P mutation decreased kinetic stabil- ity and enhanced thermodynamic stability [30]. In another study, http://dx.doi.org/10.1016/j.ijbiomac.2017.03.069 0141-8130/© 2017 Elsevier B.V. All rights reserved.