Virtual design for the thermostability enhancement of ketoreductase CgKR1 based on residue interaction network...

The ketoreductase CgKR1-F92C/F94W from Candida glabrata has high catalytic activity and stereoselectivity to efficiently synthesize the key chiral intermediate (S)-N-Boc-3-hydroxypiperidine for the anticancer drug ibrutinib. However the unsatisfying thermostability limits its practical application. In this study, rational modification strategies were constructed based on molecular simulations to improve the thermostability of CgKR1. Firstly, beneficial mutation M₁ (D138Q) and M₂ (D138Q/H276C/Q30M) were identified based on sequence analysis and unfolding free energy computational methods. The half-life of M₂ measured at 50 °C was increased by 31.5-fold compared to that of the parent. The RINVES (Residue Interaction Network-based Virtual Design for Enzyme Stability) strategy was then constructed to convert the structure of the protein into residue interaction network. Topology parameters of the networks were analyzed to identify the potential mutants with higher thermostability. A dominant mutation M₃ (D138Q/H276C/Q30M/A313R/A302R) was obtained using this strategy, with a 62.3-fold increase in t_1/2 at 50 °C and a 13 °C increase in T₅₀¹⁵ compared to the parent, while maintaining a high level of catalytic activity. Molecular dynamics simulation results showed that the mutated residues introduced new interactions with surrounding amino acids, which may contributed to the enhanced thermostability. Finally, the M₃ mutant demonstrates efficient asymmetric reduction capabilities, achieving 95% conversion of 100 g L^-1 the substrate N-Boc-3-piperidone after 4 h, whereas the conversion was only 56% in the parent due to premature inactivation. Our work not only contributes promising biocatalyst for the biosynthesis of (S)-N-Boc-3-hydroxypiperidine, but also offers a new alternative strategy for efficient enzyme thermostability engineering.