title: Molecular basis of thermostability enhancement of Renilla luciferase at higher temperatures by insertion of a disulfide bridge into the structure.
abstract: Renilla luciferase (RLuc), also known as Renilla-luciferin 2-monooxygenase, is a light producing enzyme used in many biotechnological applications such as bioreporters. However, its kinetics stability -especially at higher temperatures- is a limiting factor for developing thermostable bioreporters. The aim of this study was to improve the stability of super Renilla luciferase 8 (SRLuc 8) which is a red-emitter variety of RLuc at higher temperatures, by introduction of a disulfide bridge into its structure. In this study, the choice of the proper disulfide bond formation was based on computational methods and enzyme functionality (active site position) which is called geometric-functional method. N45 and A71 at the N-terminal of the enzyme were selected for directed evolution. The engineered luciferase was called C-SRLuc 8 and its activity and stability were assayed. The results indicated that the kinetic stability of C-SRLuc 8 increased significantly at 60°C to 70°C as compared to SRLuc 8; the residual activity of C-SRLuc 8 was approximately 20% after incubation at 65°C for 5min. Moreover, the enzyme activity decreased compared with SRLuc 8. The molecular basis of the structural changes was considered using molecular dynamics simulations and the results indicated that the N45C/A71C crosslink was involved in a hotspot foldon which seemed to be the rate-limiting step of conformational collapse at higher temperatures. The present study may provide an opportunity for the development of the next-generation of thermostable RLuc-based biosensors.
authors.affiliation: Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Fanaei Kahrani
authors.affiliation: Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.. Electronic address: Sci_rahman@yahoo.com.
authors.forename: Rahman
authors.initials: R
authors.lastname: Emamzadeh
authors.affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Mahboobeh
authors.initials: M
authors.lastname: Nazari
authors.affiliation: Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.
authors.forename: Seyed Mohammad Mahdi
authors.initials: SMM
authors.lastname: Rasa
mesh_terms: D001709:Biotechnology / Q000379:methods; D020134:Catalytic Domain / Q000502:physiology; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability / Q000502:physiology; D006358:Hot Temperature; D007700:Kinetics; D008027:Light; D049410:Luciferases, Renilla / Q000737:chemistry* / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed / Q000379:methods; D013696:Temperature
chemical_list: D004220:Disulfides; D049410:Luciferases, Renilla
keywords: Disulfide bridge; Kinetic stability; Molecular dynamic simulation; Super Renilla luciferase 8 (SRLuc 8)