Counteraction of stability-activity trade-off of Nattokinase through flexible region shifting

The widespread trade-off between stability and activity severely limits enzyme evolution. Although some pro-gresses 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 coun-teracting 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.

Automated summary

A combinatorial mutant M4 with improved stability and activity was obtained through multi-strategy engineering, showing a 20.7-fold increase in half-life and doubled catalytic efficiency. Molecular dynamics simulation revealed an obvious flexible region shifting in the structure of mutant M4, which was considered as a key factor in counteracting the stability-activity trade-off. Further analysis showed that the flexible region shifting was driven by region dynamical networks reshaping. This work provides insights into the counteraction mechanism and offers a potential strategy for enzyme evolution through computational protein engineering.