Developing hybrid systems to address oxygen uncoupling in multi-component Rieske oxygenases.

Area of Science:

• Biochemistry
• Enzymology
• Biocatalysis

Background:

• Rieske non-heme iron oxygenases (ROs) are crucial redox enzymes for biodegradation and natural product synthesis.
• Their oxygen activation mechanism involves an electron transfer chain (ETC), which risks reactive oxygen species (ROS) formation.
• Previous work linked ROS to O2 uncoupling in cumene dioxygenase (CDO) reductase.

Purpose of the Study:

• To investigate the impact of ROS formation on the multi-component CDO system in a cell-free environment.
• To assess the effects of hydrogen peroxide (H2O2) on CDO's in vitro catalytic efficiency with a NADH cofactor regeneration system.

Main Methods:

• Investigated ROS formation and its effect on CDO activity in vitro.
• Evaluated hybrid systems with alternative redox partners for CDO.
• Compared product formation and ROS generation between native and hybrid systems.

Main Results:

• ROS formation, specifically H2O2, negatively impacts CDO's catalytic efficiency.
• Hybrid systems with alternative redox partners significantly reduce H2O2 formation.
• A hybrid system using phthalate dioxygenase reductase (PDR) with CDO enhanced indene oxyfunctionalization 4-fold.

Conclusions:

• Hybrid biocatalytic systems offer a strategy to mitigate ROS formation and improve enzyme efficiency.
• Alternative redox partners are advantageous for reducing H2O2 and increasing product yield in RO-based biocatalysis.