Custom tuning of Rieske oxygenase reactivity

Rieske oxygenases use a Rieske-type [2Fe-2S] cluster and a mononuclear iron center to initiate a range of chemical transformations. However, few details exist regarding how this catalytic scaffold can be predictively tuned to catalyze divergent reactions. Therefore, in this work, using a combination of structural analyses, as well as substrate and rational protein-based engineering campaigns, we elucidate the architectural trends that govern catalytic outcome in the Rieske monooxygenase TsaM. We identify structural features that permit a substrate to be functionalized by TsaM and pinpoint active-site residues that can be targeted to manipulate reactivity. Exploiting these findings allowed for custom tuning of TsaM reactivity: substrates are identified that support divergent TsaM-catalyzed reactions and variants are created that exclusively catalyze dioxygenation or sequential monooxygenation chemistry. Importantly, we further leverage these trends to tune the reactivity of additional monooxygenase and dioxygenase enzymes, and thereby provide strategies to custom tune Rieske oxygenase reaction outcomes. Rieske oxygenase chemistry is important for biochemical pathways, but it remains elusive how a common protein scaffold can be predictively tuned to catalyze divergent reactions. Here, the authors report a strategy that can rationally tune TsaM, a Rieske monooxygenase to catalyze dioxygenation and sequential monooxygenation reactions, and customize the reactivity of other Rieske oxygenases.

Automated summary

By combining structural analyses, substrate and rational protein-based engineering, this study elucidates the architectural trends that govern catalytic outcome in the Rieske monooxygenase TsaM and provides strategies to custom tune Rieske oxygenase reaction outcomes.