Photosensitizer Repositioning Affords an Enantiocomplementary Enzyme for [2 + 2]-Cycloadditions.

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Chemistry

Background:

• Enantioselective photoenzymes enable [2+2]-cycloadditions of quinolone and indole derivatives.
• Existing enzymes yield only one enantiomeric series of cyclobutane products.

Purpose of the Study:

• To develop an enantiocomplementary [2+2]-cyclase by repositioning a photosensitizer.
• To engineer a highly selective and oxygen-tolerant photoenzyme for cycloaddition reactions.

Main Methods:

• Genetic code expansion and directed evolution.
• Crystal structure analysis of engineered enzyme EnT1.3.
• Molecular dynamic simulations of the CEnT1.4 active site.

Main Results:

• A new enantiocomplementary photoenzyme, CEnT1.0, was created by repositioning the photosensitizer.
• Directed evolution yielded CEnT1.4, a proficient and oxygen-tolerant enzyme.
• CEnT1.4 achieved 99% enantioselectivity and improved regioselectivity (62:1) for quinolone cycloadditions.

Conclusions:

• Genetically programmed photosensitizers offer versatility for altering enzyme stereochemical outcomes.
• The engineered active site pocket pre-organizes substrates for selective catalysis.
• This work expands the scope of biocatalytic cycloaddition reactions.