Improving the activity of rieske dioxygenases for benzoates through enzyme engineering

Abstract

Rieske dioxygenases (RDOs) are well-known oxidoreductase enzymes commonly found in soil bacteria. They participate in the metabolism of aromatic pollutants in their environment, and their utility in organic synthesis has been noted due to RDOs ability to catalyze the asymmetric dihydroxylation of aromatic compounds to form chiral cis-diene-diol metabolites. RDOs are limited as green-chemical catalysts, however, because of steric and electronic constraints within their active sites that lead to restricted substrate scopes and minimal activity for specific substrate classes. This research consisted of using active site-targeted mutagenesis alongside a recently developed high throughput assay system to develop novel toluene dioxygenase (TDO) variants with appreciably increased activity in the dihydroxylation of esterfunctionalized substrates, specifically methyl and ethyl benzoate, in comparison to the native enzyme. Eight distinct point mutations were found to give improved activity for the chosen substrates, including two novel mutations (I276C and I276T) that no other study has previously identified as being beneficial. The activity level of these variants increased upon the native enzyme’s activity for methyl benzoate by up to 594%, 590%, and 494% for I276V, M220A, and L272W respectively. Likewise, the activity of variants M220A, I276C, and I276V were 9363%, 5461%, and 5342% respectively for ethyl benzoate compared to the unaltered TDO system. Characterization of these variants has developed a library of beneficial mutations which will guide the generation of enzyme variants with multiple substitutions to investigate potential synergistic effects on activity and substrate scope. This study will serve to inform future engineering studies targeting RDOs, thus broadening the utility of these environmentally friendly catalysts as green-chemical tools for the creation of vital building blocks in organic synthesis.