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.

Exploring the Potential of Arginine to Increase Coelenterazine-Renilla Luciferase Affinity and Enzyme Stability: Kinetic and Molecular Dynamics Studies

“…Therfore, Renilla Luciferase has been used in biotechnology applications such as molecular imaging and gene reporters [7][8][9][10]. However, the applications of the enzyme are limited due to the low thermal stability of this enzyme, and so far efforts have been made to improve the stability of the enzyme by adding additives or by site-directed mutagenesis studies [6,[11][12][13][14].…”

New insights into the molecular characteristics behind the function of Renilla luciferase

“…The data obtained from the crystallization of RLuc8 and other experiments suggest that the enzyme contains 311 residues and has a molecular weight of approximately 37 kDa . Moreover, it has a deep tunnel made up of mostly hydrophobic residues and the catalytic triad (D120, E144, and H285) is embedded at the bottom of the tunnel . However, there is no crystallographic evidence of the RLuc‐coelenterazine‐h complex, because its crystallization requires specific and anoxic condition.…”