Rational Side-Chain Amino Acid Substitution in Firefly Luciferase for Improved Thermostability

Improving the thermostability of the luciferase from firefly (Photinus pyralis) needs to be solved to broaden its industrial applications. In this paper, molecular dynamic (MD) simulations were used to identify 4 amino acid substitutions (P183V, E325K, Q338V, and E354K) which might have a significant influence on the thermostability of luciferase. Root-mean-square deviation values were calculated to further evaluate the effect of these mutations on thermostability of the enzyme and demonstrated that the thermostability of the corresponding protein variants was in the order E354K > E325K > WT > P183V > Q338V. Following the MD simulation, the enzyme variants were expressed in a recombinant host, and the results showed that the t1/2, T50, and Tm of mutant E354K were increased 2.32-fold, and 4.5 and 3.3°C more compared with the wild type, respectively. MD simulations, as well as circular dichroism and fluorescence spectroscopy were further applied to elucidate the conformational differences between the wild-type and E354K luciferases. The results indicated that a possible explanation for the improved thermostability of E354K enzyme lies in the formation of a salt bridge between Lys354 and Glu311 and alteration of protein conformation. Firefly luciferases catalyzes the oxidation of d-luciferin in the presence of Mg2+-ATP and oxygen, accompanied by a high quantum yield of bioluminescence [1, 2]. This characteristic is the main reason for its wide use in molecular biology, food hygiene, and medicine, especially for the detection of microorganisms [3–5]. However, many practical applications of luciferase-based assays are hindered by the enzyme’s poor thermostability at elevated temperatures. Structural features of the molecule are known to play an important role in enzyme stability. Khajeh et al. used site-directed point mutations in Bacillus to obtain 2 laccase variants which showed higher stability against urea as a chemical denaturant [6]. Mishra et al. obtained a thermostable mutant of the lipase from Bacillus licheniformis RSP-09 by two rounds of directed evolution [7], and it showed a 13.5-fold increase in thermostability at 60°C for 60 min over the wild-type lipase. However, not all mutations play a driving role in boosting the thermostability of enzymes. Usually, large numbers of experiments are needed, which is time- and labor-consuming. Therefore, rational design strategies to improve enzyme thermostability are urgently needed. Reports show that thermostable enzymes are more rigid, have lower root-mean-square deviation (RMSD) and are compact (represented by the radius of gyration) [8], which could be simulated based on molecular dynamics simulations (MD). MD simulations have been thought as a more effective rational design strategy for the evaluation of enzymatic stability effects [9, 10]. For example, Tian et al. generated the G149P mutant of methyl parathion hydrolase with a lower RMSD value, which was found to possess higher thermostability than the native enzyme [11]. Zhu et al. used RMSD values to evaluate the thermostability of mutants and obtained the thermostable TLM2 mutant of thermolysin, which showed a 3.1-fold longer half-life than the wild-type at the same temperature [12]. In this study, a rational design strategy under the guidance of MD was used to select and identify target site effects to improve the thermal stability of firefly luciferase. The obtained mutant E354K showed improved thermostability without lowering the catalytic activity. Furthermore, thermoduric mechanisms underlying this substitution were investigated via a detailed analysis of the resulting structural changes.