Reversed Enantiopreference of an ω‐Transaminase by a Single‐Point Mutation

Abstract Altering the characteristics of an active‐site loop in an ( S )‐selective ω‐transaminase from Arthrobacter citreus (variant CNB05‐01) influences the enantioselectivity. This active‐site loop belongs to the second subunit of the dimeric enzyme structure that participates in the coordination of pyridoxal‐5′‐phosphate (PLP) in the so called “phosphate group binding cup”. Three amino acid residues (E326, V328, and Y331) in this loop are selected by homology modeling for site‐directed mutagenesis aiming to increase the enzyme enantioselectivity for 4‐fluorophenylacetone. By combining these mutations, five enzyme variants are created. The performance of these variants is explored using a model system consisting of isopropylamine and 4‐fluorophenylacetone or 4‐nitroacetophenone in asymmetric synthesis using a whole‐cell system approach. Three of the five variants show increased enantioselectivity for 4‐fluorophenylacetone compared to CNB05‐01. Variant CNB05‐01/Y331C increases the enantioselectivity from 98 % ee to over 99.5 % ee . A single‐point mutation, V328A, turn the ( S )‐selective ω‐transaminase into an ( R )‐selective enzyme. This switch in enantioselectivity is substrate dependent, exhibiting ( R ) selectivity for 4‐fluorophenylacetone and retaining ( S ) selectivity for 4‐nitroacetophenone. The shift in enantiopreference is further confirmed by molecular docking simulations. Homology modeling is shown to be a powerful tool to target important amino acid residues in this enzyme in order to improve enantioselectivity by rational design.