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Rieske dioxygenases are multi-component enzyme systems, naturally found in many soil bacteria, that have been widely applied in the production of fine chemicals, owing to the unique and valuable oxidative dearomatization reactions they catalyze. The range of practical applications for these enzymes in this context has historically been limited, however, due to their limited substrate scope and strict selectivity. To overcome these limitations, our research group has employed the tools of enzyme engineering to expand the substrate scope or improve the reactivity of these enzyme systems in specific contexts. Traditionally, enzyme engineering campaigns targeting metalloenzymes have avoided mutations to metal-coordinating residues, based on the assumption that these residues are essential for enzyme activity. Inspired by the success of other recent enzyme engineering reports, our research group investigated the potential to alter or improve the reactivity of Rieske dioxygenases by altering or eliminating iron coordination in the active site of these enzymes. Herein, we report the modification of all three iron-coordinating residues in the active site of toluene dioxygenase both to alternate residues capable of coordinating iron, and to a residue that would eliminate iron coordination. The enzyme variants produced in this way were tested for their activity in the czs-dihydroxylation of a small library of potential aromatic substrates. The results of these studies demonstrated that all three iron-coordinating residues, in their natural state, are essential for enzyme activity in toluene dioxygenase, as the introduction of any mutations at these sites resulted in a complete loss of czs-dihydroxylation activity for all substrates tested. [ABSTRACT FROM AUTHOR]

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 the cis-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 the cis-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., (© 2024 Wiley Periodicals LLC.)