Rieske dioxygenase (RDOs), a class of enzyme systems found in soil bacteria, play an important role in the bacterial metabolism of aromatic “pollutants” in their environment. RDOs have a long been utilized in organic synthesis due to their ability to catalyze the asymmetric dihydroxylation of aromatics to produce chiral diene-diol metabolites. Despite their utility, the range of potential applications for these green catalysts has been limited by steric and electronic constraints on their substrate scopes and on their activity.<br/><br/>Our work in the Ball State Laboratory for Biocatalysis Research is focused on the development of new green-chemical tools, in the form of engineered enzymes that operate in aqueous solutions, to help contribute to attenuating the reliance of chemical industry on non-renewable energy sources. To this end, we have applied active site targeted mutagenesis along with a recently reported high throughput screening platform to develop engineered toluene dioxygenase (TDO) variants with significantly increased activity in the dihydroxylation of a broader range of aromatic substrates. This research has led to the identification of a series of dioxygenase variants that demonstrate significantly improved activity for the dihydroxylation of ester-functionalized substrates over the parent enzyme. These variants have been characterized, which has afforded the development of an index of beneficial substitutions in this context. Further, we have iteratively combined these identified beneficial mutations to determine whether any additional synergistic effects on the enzyme activity can be obtained in this way. We aim to apply the knowledge gained in this study in engineering more improved Rieske dioxygenase variants, to broaden the utility of these environmentally sustainable catalysts.

Investigation of the effects of targeted active site mutations on the activity of Rieske dioxygenases

ACS Spring 2022

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