Light-driven biocatalytic oxidation and reduction reactions: scope and limitations

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Photochemistry

Background:

• Nicotinamide-dependent oxidoreductases are crucial biocatalysts.
• Developing practical regeneration systems for these enzymes remains a challenge.
• Visible light offers a sustainable approach for enzyme regeneration.

Purpose of the Study:

• Investigate limitations in light-driven regeneration of flavin-dependent monooxygenase (PAMO-P3).
• Identify factors affecting catalytic performance and turnover frequency.
• Extend light-driven regeneration to other flavin-dependent enzymes, like Old Yellow Enzyme homologue YqjM.

Main Methods:

• Utilized visible light for direct reductive regeneration of PAMO-P3.
• Performed light-driven enantioselective Baeyer-Villiger oxidations.
• Analyzed electron transfer kinetics and identified oxidative uncoupling using EDTA as electron donor.
• Applied the light-driven regeneration approach to YqjM.

Main Results:

• Oxidative uncoupling led to ~95% loss of reducing equivalents.
• Turnover frequency for PAMO-P3 was two orders of magnitude lower than conventional methods.
• Sluggish electron transfer kinetics due to impeded flavin-catalyst interaction were identified as a bottleneck.
• Significantly higher catalytic turnover was achieved with YqjM, attributed to better cofactor accessibility and no oxidative uncoupling.

Conclusions:

• Oxidative uncoupling and slow electron transfer kinetics limit the efficiency of light-driven regeneration for PAMO-P3.
• Optimizing cofactor accessibility and minimizing uncoupling are crucial for effective biocatalysis.
• Light-driven regeneration is a viable strategy for other flavin-dependent reductases, showing improved performance in model systems.