Extended Data Fig. 3: Directed evolution of an efficient and enantioselective photoenzyme.

Schematic showing the trajectory from DA_20_00 to EnT1.3 and EnT1.3 Y121F S271C. Mutations introduced are represented as CPK spheres at the C-alpha. The original design DA_20_00 contains 13 mutations built into a six-bladed β-propeller scaffold (PDB 1E1A; a diisopropylfluorophosphatase from Loligo vulgaris)¹³. Incorporation of the photosensitizer BpA (green atom-coloured sticks and semitransparent CPK spheres) at position A173 provided EnT1.0. An alanine scan of EnT1.0 highlighted one variant with increased activity, providing EnT1.1. Two rounds of evolution afforded EnT1.3, which contains six mutations when compared with the original design DA_20_00. EnT1.3 was further evolved towards substrate 13, resulting in EnT1.3 Y121F S271C. Library generation method, positions targeted, the number of clones evaluated, beneficial mutations and the most improved variant for each round are given in the table.