Rational selection of circular permutation sites in characteristic regions of the α/β-hydrolase fold enzyme RhEst1

• Rationally defining the characteristic regions for selection of circular permuted sites was firstly proposed.
• We successfully designed the longest linker for circular permutation of the α/β-hydrolase.
• Improved the thermostability of RhEst1 by circular permutation.
• We summarized the general guidelines for circular permutation of the α/β-hydrolase fold enzyme.

Circular permutation (CP) involves the cleavage of polypeptide to obtain new termini, which can result in different protein structures and functions. It has been demonstrated to be an effective strategy for the evolution of proteins, but the lack of principle for selecting CP site to construct functional variants is still a challenge for CP. In this study we performed the CP analysis of the typical esterase RhEst1 to explore the CP site-selection strategy of the α/β-hydrolase fold family. A CP library of 97 mutants was generated to identify the effect of CP on three characteristic regions of RhEst1 including the flexible cap domain (Region 1), the region around the entrance to substrate binding pocket (Region 2) and the surface-exposed sectors in catalytic domain (Region 3). We found the protein folding, stability and bioactivity of CP variants were altered significantly and the CP sites of active variants were mainly located in the flexible loops. These studies reveal the importance of site-selection for CP and provide more information for CP of other α/β-hydrolases.

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