Protein termini relocation plus random mutation: A new strategy for finding key sites in esterase evolution

Abstract

A pharmaceutically relevant esterase, RhEst1, could catalyze the hydrolysis of (R,S)-ethyl-2,2-dimethyl cyclopropane carboxylate [(R,S)-DmCpCe] with excellent enantioselectivity, producing (S)-(+)-2,2-dimethyl cyclopropane carboxylic acid [(S)-DmCpCa], which is a key chiral building block for the synthesis of Cilastatin. In our previous work, a mutant RhEst1-M2 was identified with 6.4-fold higher activity than the wild-type. Additionally, the termini of RhEst1 protein were altered by circular permutation (CP), resulting in a mutant CP-176 which still maintains the catalytic activity of esterase. In this work, to improve the catalytic properties of RhEst1, the mutant CP-176 was taken as the parent of directed evolution. Consequently, a new mutant designated as CP-M1 (=CP-176G282S) was identified, indicating 3.2-fold catalytic efficiency enhancement and nearly 7 ℃ improvement in melting temperature (Tm) as compared with CP-176. Furthermore, the beneficial mutation “G282S” of CP-M1 was reversely introduced into RhEst1-M2, generating the best mutant M3 (=RhEst1-M2G167S), with 1.8-fold catalytic efficiency improvement and nearly 10 ℃ improvement of Tm, as compared with RhEst1-M2. This is the first report that the circular permutation and random mutagenesis were combined to reshape a protein, affording distinctly improved activity and thermostability.