Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C-C Bond-Forming Enzyme

Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem-fused 4-OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4-OT was then subjected to eleven rounds of directed evolution to give variant 4-OT(F11), which showed an up to 320-fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4-OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180 degrees relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins.

Automated summary

This study used gene duplication and fusion strategy to evolve a promiscuous homohexameric enzyme into an efficient catalyst with high catalytic activity for enantioselective reactions. Through directed evolution and structural analysis, the asymmetric features of the protein were revealed, suggesting that this strategy may become an important technique in enzyme engineering.