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 tandem-fused 4-OT allow independent sequence diversification of adjacent subunits by directed evolution. This fused was then subjected eleven rounds evolution give variant 4-OT(F11), which showed up 320-fold enhanced activity addition nitromethane cinnamaldehydes. Crystallographic analysis revealed 4-OT(F11) has unusual asymmetric trimeric architecture in one monomers is flipped 180° relative others. gene break structural symmetry likely become indispensable asset enzyme engineering toolbox, finding wide use oligomeric proteins.