Photoenzymes are biological catalysts that use light to convert starting materials into products. These catalysts require photon absorption for each turnover, making quantum efficiency an important optimization parameter. Flavin-dependent "ene"-reductases (EREDs) display latent photoenzymatic activity for synthetically valuable hydroalkylations; however, protein engineering has not been used to optimize this nonnatural function. We describe a protein engineering platform for the high throughput optimization of photoenzymes. A single round of engineering results in improved catalytic function toward the synthesis of g, d, e-lactams, and acyclic amides. Mechanistic studies show that key mutations can alter the enzymes excited state dynamics, enhance its photon efficiency, and ultimately increase catalyst performance. Transient absorption spectroscopy reveals that engineered variants display dramatically decreased radical lifetimes, indicating an evolution toward a concerted mechanism.

Biocatalysis has revolutionized chemical synthesis, providing sustainable methods for preparing various organic molecules. In enzyme-mediated organic synthesis, most reactions involve molecules operating from their ground states. Over the past 25 years, there has been an increased interest in enzymatic processes that utilize electronically excited states accessed through photoexcitation. These photobiocatalytic processes involve a diverse array of reaction mechanisms that are complementary to one another. This comprehensive review will describe the state-of-the-art strategies in photobiocatalysis for organic synthesis until December 2022. Apart from reviewing the relevant literature, a central goal of this review is to delineate the mechanistic differences between the general strategies employed in the field. We will organize this review based on the relationship between the photochemical step and the enzymatic transformations. The review will include mechanistic studies, substrate scopes, and protein optimization strategies. By clearly defining mechanisticallydistinct strategies in photobiocatalytic chemistry, we hope to illuminate future synthetic opportunities in the area.

Photoenzymes are biological catalysts that use light to convert starting materials into products. These catalysts require photon absorption for each turnover, making quantum efficiency an important optimization parameter. Flavin-dependent "ene"-reductases (EREDs) display latent photoenzymatic activity for synthetically valuable hydroalkylations; however, protein engineering has not been used to optimize this nonnatural function. We describe a protein engineering platform for the high throughput optimization of photoenzymes. A single round of engineering results in improved catalytic function toward the synthesis of g, d, e-lactams, and acyclic amides. Mechanistic studies show that key mutations can alter the enzymes excited state dynamics, enhance its photon efficiency, and ultimately increase catalyst performance. Transient absorption spectroscopy reveals that engineered variants display dramatically decreased radical lifetimes, indicating an evolution toward a concerted mechanism.