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Abstract Rieske dioxygenases have a long history of being utilized as green chemical tools in the organic synthesis of high‐value compounds, due to their capacity to perform thecis‐dihydroxylation of a wide variety of aromatic substrates. The practical utility of these enzymes has been hampered however by steric and electronic constraints on their substrate scopes, resulting in limited reactivity with certain substrate classes. Herein, we report the engineering of a widely used member of the Rieske dioxygenase class of enzymes, toluene dioxygenase (TDO), to produce improved variants with greatly increased activity for thecis‐dihydroxylation of benzoates. Through rational mutagenesis and screening, TDO variants with substantially improved activity over the wild‐type enzyme were identified. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in an effort to elucidate how the identified mutations resulted in improved activity for this polar substrate class. These analyses revealed modification of the substrate tunnel as the likely cause of the improved activity observed with the best‐performing enzyme variants. 

Rational engineering of toluene dioxygenase expands the substrate scope of this enzyme, enabling the production of new, amide-functionalized chiral metabolites.