Engineering a Non-Natural Photoenzyme for Improved Photon Efficiency.

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Photochemistry

Background:

• Photoenzymes utilize light for catalysis, with quantum efficiency being crucial.
• Flavin-dependent ene-reductases (EREDs) possess latent photoenzymatic activity for hydroalkylation reactions.
• Optimization of EREDs for this non-natural function via protein engineering is underexplored.

Purpose of the Study:

• To develop a high-throughput protein engineering platform for optimizing photoenzymes.
• To enhance the catalytic efficiency of flavin-dependent ene-reductases for hydroalkylation.
• To investigate the mechanistic basis for improved photoenzymatic activity.

Main Methods:

• High-throughput screening of engineered photoenzymes.
• Protein engineering and directed evolution approaches.
• Transient absorption spectroscopy to study excited state dynamics.

Main Results:

• A single round of protein engineering significantly improved catalytic function.
• Engineered variants demonstrated enhanced synthesis of γ, δ, ϵ-lactams and acyclic amides.
• Mutations altered excited state dynamics, increased photon efficiency, and reduced radical lifetimes.

Conclusions:

• A novel protein engineering platform effectively optimizes photoenzymes.
• Engineered EREDs exhibit superior performance in light-driven hydroalkylation.
• Mechanistic insights reveal a shift towards a concerted reaction mechanism in improved variants.