Photosensitizer Repositioning Affords an Enantiocomplementary Enzyme for [2 + 2]‐Cycloadditions

Abstract The combination of genetic code expansion and directed evolution has recently given rise to enantioselective photoenzymes for [2 + 2]‐cycloadditions of quinolone and indole derivatives. However, the enzymes reported to date only allow access to one enantiomeric series of the strained cyclobutane products. Here, guided by a crystal structure of our previously engineered enzyme EnT1.3, we show how judicious repositioning of the genetically programmed benzophenone photosensitizer affords an enantiocomplementary [2 + 2]‐cyclase, CEnT1.0. Following directed evolution, a proficient and oxygen‐tolerant photoenzyme (CEnT1.4) emerged that promotes [2 + 2]‐cycloadditions of a quinolone derivative with exquisite enantiocontrol (99% e.e .) and substantially enhanced regioselectivity compared with EnT1.3 (r.r. 62:1 vs. 9:1). Structural analysis of CEnT1.4, coupled with molecular dynamic simulations, reveals a well‐sculpted active site pocket that pre‐organises the substrate for regio‐ and enantioselective catalysis. This study highlights the versatility offered by genetically programmed (photo)catalytic elements when developing enzymes for altered stereochemical outcomes.