Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM

Rieske nonheme iron oxygenases use two metallocenters,a Rieske-type[2Fe-2S] cluster and a mononuclear iron center, to catalyze oxidationreactions on a broad range of substrates. These enzymes are widelyused by microorganisms to degrade environmental pollutants and tobuild complexity in a myriad of biosynthetic pathways that are industriallyinteresting. However, despite the value of this chemistry, there isa dearth of understanding regarding the structure-functionrelationships in this enzyme class, which limits our ability to rationallyredesign, optimize, and ultimately exploit the chemistry of theseenzymes. Therefore, in this work, by leveraging a combination of availablestructural information and state-of-the-art protein modeling tools,we show that three hotspot regions can be targetedto alter the site selectivity, substrate preference, and substratescope of the Rieske oxygenase p-toluenesulfonatemethyl monooxygenase (TsaM). Through mutation of six to 10 residuesdistributed between three protein regions, TsaM was engineered tobehave as either vanillate monooxygenase (VanA) or dicamba monooxygenase(DdmC). This engineering feat means that TsaM was rationally engineeredto catalyze an oxidation reaction at the meta and ortho positions of an aromatic substrate, rather than itsfavored native para position, and that TsaM was redesignedto perform chemistry on dicamba, a substrate that is not nativelyaccepted by the enzyme. This work thus contributes to unlocking ourunderstanding of structure-function relationships in the Rieskeoxygenase enzyme class and expands foundational principles for futureengineering of these metalloenzymes.

Automated summary

Rieske nonheme iron oxygenases utilize two metallocenters to catalyze oxidation reactions on various substrates. Understanding the structure-function relationships in this enzyme class is crucial for rational redesign and optimization of these enzymes. In this study, by using available structural information and protein modeling tools, the researchers successfully engineered the Rieske oxygenase TsaM to behave as different monooxygenases and expand its substrate range.