Thermostabilization and molecular dynamics simulation of NaStyA for highly enantioselective (R)-epoxidation of styrene analogues

Abstract

Chiral epoxides are one of the most versatile building blocks in organic synthesis, but the asymmetric epoxidation of unfunctionalized terminal olefins remains a challenge. NaStyA is a styrene monooxygenase from Nocardia altamirensis that functions as an enantioselective epoxygenase for terminal olefins. To improve its robustness, we investigated over 50 mutants designed via evolutionary- and energy-based approaches, and successfully identified 18 beneficial mutations, which then led to a combinatorial mutant of TM18 with drastically enhanced thermostability and solvent tolerance while retaining excellent activity and enantioselectivity. Its turnover frequency and half-life at 50 °C were increased to 5- and 148-fold of the parental. In the epoxidation of terminal olefins, TM18 achieved high product yields of over 4-fold of the parental, and excellent enantiomeric excess of 99 % to >99 % ee. The analysis of trajectory data from molecular dynamics simulations further validated that the mutations in TM18 increased its local rigidity, as inferred by RMSF and PCA analyses and through novel salt bridges formation. Furthermore, TM18 displayed reduced residue cross-correlation motions. The combined effects of these mutations result in an overall improvement in the structural and dynamic thermostability of TM18.