Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

The flexible regions represented by loops have been the focus of protein stability engineering. However, residues located in specific rigid regions are also crucial for protein stability. The role of dynamic correlations between mutations in these two regions on protein stabilization remains inadequately understood. In this work, a ketoreductase CpKR originated from Candida parapsilosis was rationally engineered to improve its stability by comprehensively redesigning the flexible and rigid regions. Hot spots were successively identified and validated in the rigid regions with low B-factor or root-mean-square fluctuation values, as well as in relatively flexible areas of the protein. Based on the non-additive and cooperative mutational effects, a combinatorial variant (M2) with beneficial mutations in these two distinct regions showed a 4630-fold increased half-life at 40 °C and a 12 °C higher apparent melting temperature as compared with those of the wild type. The variant M2 also showed improved pH compatibility, better organic substance tolerance, and enhanced performance in asymmetric reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (1a), the precursor of cardiovascular drug Diltiazem. The crystal structures and molecular dynamics simulations demonstrated that additional interaction networks in both the rigid and flexible regions modulated protein stability, and the dynamic correlation between these two regions mediated the observed synergistic combination. These results showed that both the flexible and rigid regions, especially the cross-correlated areas, are crucial for enhancing protein stability, and comprehensively redesigning these two regions is a powerful and attractive approach for acquiring stable enzymes.