Expansion of the substrate scope of Toluene Dioxygenase through enzyme engineering

The Rieske dioxygenase (RDO) family of enzymes is known for its ability to stereoselectively dihydroxylate aromatic substrates, to generate chiral diene-diol metabolites, which serve as precursors in the synthesis of valuable organic compounds. However, these enzymes have so far been unable to react with a number of substrates due to the steric and/or electronic nature of the relevant compounds. In an effort to expand the utility of these enzymes, the enzyme toluene dioxygenase (TDO) was engineered to develop TDO variants that possess novel activity for amide-functionalized substrates. This was performed by identifying active site residues for mutation, creating a series of mutant libraries via directed mutagenesis, and then assessing the activity of the mutants for amide-functionalized substrates via the MPFCD assay. Putative hits that demonstrated activity for these substrates were subjected to secondary screening to confirm the activity observed upon initial screening. Confirmed hits were then genetically sequenced in order to identify the beneficial mutations, and the activity of all confirmed hits were compared in parallel.