The widespread trade-off between stability and activity severely limits enzyme evolution. Although some progresses have been made to overcome this limitation, the counteraction mechanism for enzyme stability-activity trade-off remains obscure. Here, we clarified the counteraction mechanism of the Nattokinase stability-activity trade-off. A combinatorial mutant M4 was obtained by multi-strategy engineering, exhibiting a 20.7-fold improved half-life; meanwhile, the catalytic efficiency was doubled. Molecular dynamics simulation revealed that an obvious flexible region shifting in the structure of mutant M4 was occurred. The flexible region shifting which contributed to maintain the global structural flexibility, was considered to be the key factor for counteracting the stability-activity trade-off. Further analysis illustrated that the flexible region shifting was driven by region dynamical networks reshaping. This work provided deep insight into the counteraction mechanism of enzyme stability-activity trade-off, suggesting that flexible region shifting would be an effective strategy for enzyme evolution through computational protein engineering.
Molecular Dynamics Simulation , Protein Engineering , Subtilisins/metabolism , Enzyme Stability
