Improvement of Rieske Dioxygenase Activity Through Remodeling of the Substrate Tunnel

Abstract Rieske dioxygenases are enzyme systems that have a long history of being applied as chiral, green chemical catalysts in the production of valuable building blocks for organic synthesis, owing to their ability to catalyze the cis ‐dihydroxylation of aromatics. The practical utility of these catalysts, however, has been limited by restrictions on their substrate scope and selectivity. Recent studies have demonstrated the potential of modifying the substrate tunnel of oxidase enzymes to modulate the selectivity and activity of these enzymes for specific substrates. Herein, we report the targeted modification of residues lining the substrate tunnel of a representative and widely used Rieske dioxygenase, toluene dioxygenase (TDO). Several enzyme variants generated through modification of the residues lining the substrate tunnel demonstrated substantially improved activity over the wild‐type enzyme for multiple substrates. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in efforts to elucidate how the identified mutations resulted in improved activity. These analyses suggested that new interactions introduced along the substrate tunnel may explain the improved activity observed with the best‐performing enzyme variants.