Engineering Rieske oxygenase activity one piece at a time

Enzyme engineering plays a central role in the development of biocatalysts for biotechnology, chemical and pharmaceutical manufacturing, and environmental remediation. Rational design of proteins has historically relied on targeting active site residues to confer a protein with desirable catalytic properties. However, additional hotspots are also known to exist beyond the active site. Structural elements such as subunit-subunit interactions, entrance tunnels, and flexible loops influence enzyme catalysis and serve as potential hotspots for engineering. For the Rieske oxygenases, which use a Rieske cluster and mononuclear iron center to catalyze a challenging set of reactions, these outside of the active site regions are increasingly being shown to drive catalytic outcomes. Therefore, here, we highlight recent work on structurally characterized Rieske oxygenases that implicates architectural pieces inside and outside of the active site as key dictators of catalysis, and we suggest that these features may warrant attention in efforts aimed at Rieske oxygenase engineering.

Automated summary

Enzyme engineering is crucial for various applications, including biotechnology, chemical manufacturing, pharmaceuticals, and environmental remediation. Traditionally, protein design focused on active site residues for desired catalytic properties, but recent studies show that structural elements beyond the active site, like subunit-subunit interactions and flexible loops, also impact enzyme catalysis. This is particularly true for Rieske oxygenases, where the architectural features inside and outside the active site play a significant role in catalysis. Therefore, future Rieske oxygenase engineering efforts should consider these features.