7PUO

Structure of a fused 4-OT variant engineered for asymmetric Michael addition reactions

Summary for 7PUO

• Entry DOI: 10.2210/pdb7puo/pdb
• Descriptor: 2-hydroxymuconate tautomerase,Chains: A,B,C,D,E,F,2-hydroxymuconate tautomerase, GLYCEROL, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: protein engineering, 4-oxalocrotonate tautomerase, michael addition, carbon-carbon lyase, lyase
• Biological source: Pseudomonas putida (Arthrobacter siderocapsulatus); More
• Total number of polymer chains: 6
• Total formula weight: 82368.47

Authors

Rozeboom, H.J.,Thunnissen, A.M.W.H.,Poelarends, G.J. (deposition date: 2021-09-30, release date: 2022-01-26, Last modification date: 2024-01-31)

Primary citation

Xu, G.,Kunzendorf, A.,Crotti, M.,Rozeboom, H.J.,Thunnissen, A.W.H.,Poelarends, G.J.
Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C-C Bond-Forming Enzyme.
Angew.Chem.Int.Ed.Engl., 61:e202113970-e202113970, 2022

PubMed Abstract: 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° 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.

PubMed: 34890491
DOI: 10.1002/anie.202113970

Experimental method

X-RAY DIFFRACTION (2.35 Å)