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Publication Abstract from PubMed

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.

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C-C Bond-Forming Enzyme.,Xu G, Kunzendorf A, Crotti M, Rozeboom HJ, Thunnissen AWH, Poelarends GJ Angew Chem Int Ed Engl. 2021 Dec 10:e202113970. doi: 10.1002/anie.202113970. PMID:34890491^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.