Site‐Specific Features Guiding Effective Reversible Photocontrol of Tryptophan Synthase With the Unnatural Amino Acid AzoF

This study delivers initial guidelines for the selection of effective incorporation positions for the azobenzene‐based unnatural amino acid AzoF to facilitate the successful engineering of light‐sensitive enzymes, dubbed photoxenases. Although photoxenases gain interest for the photocontrol of enzymatic activity across various fields their design has so far remained challenging. To ease particularly the selection of AzoF incorporation sites, we have systematically evaluated a total of 85 positions that differ in their biochemical and biophysical features in the bi‐enzyme model system tryptophan synthase. Medium‐throughput production of AzoF‐containing variants, activity‐based screening and evaluation of photocontrol, and subsequent validation of the screening results allowed us to identify three site‐specific features that promote photocontrol with a success rate of up to 83% when combined. These features include positions in domains with catalytically important conformational transitions, positions determined via molecular dynamics based shortest path map analysis, and positions that originally contain a hydrophilic side chain. Ultimately, our results underscore the complexity of predicting suitable incorporation sites but simultaneously provide a fundamental framework for the improved rational design of future photoxenases with AzoF.