He Huang,Meng Si, Qing Xu and Ling Jiang
School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China
Due to the substrate specificity and high yield of bioluminescence, firefly luciferase is widely used in molecular biology, food hygiene and medicine, in particular for the detection of microorganisms. However, the poor thermostability of luciferase limits their commercial applications. Molecular dynamics (MD) simulations is considered as a useful tool to discover the effect of mutation on enzyme's dynamic stability and structure, further for evaluation of enzymatic stability effect. To improve the thermostability of luciferase, we addressed the wide-type luciferase and three potential mutated systems E325K, E354K and L17A in 10-ns MD simulations at 300 K. By comparing the RMSD value and radius of gyration, we found the thermal insensitivity of variants were E354K> E325K> WT> L17A. To verify computational prediction, we conducted recombinant experiment of aforementioned mutants by site-direct mutagenesis, the result showed that the mutation had little effect on enzyme expression and activity. To investigate the thermostability of luciferase from wide-type and mutants, purified enzymes were incubated at 37 oC for 5 h, the mutant of E354K showed a higher thermostability with nearly 30% residual activity, while the native-type enzyme and other mutants showed reduced thermostability lower than 8% residual activity. Further, the half-life of E354K showed 2.6-fold of the wild-type, the T5010 and Tm of E354K were 4.5 oC and 3.2 oC higher, respectively, than that of the wide-type enzyme. These in vitro experiments strongly validated the above prediction, that E354K mutant displayed superior thermal stability. To further find the conformational differences between the native-type and E354K luciferase, CD and fluorescence spectroscopy were applied. The CD results showed a decrease in negative ellipticity at 208 and 222nm of E354K, indicated that E354K mutant contained an increased percentage of α-helical and a decreased content of β-sheet. The fluorescence spectroscopy analysis showed a decreased fluorescence intensity of E354K mutant compared with the wild-type, indicated displacement of tryptophan residues to a more hydrophilic environment, which was consistent with the computational predictions.
In summary, we have successfully improved the thermostability of luciferase by site-direct mutagenesis under the guidance of molecular dynamics simulation. The stabilization of luciferase would broad the industrial applications of this enzyme.
Keywords: Thermostability, molecular dynamic simulation, luciferase.