Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Synthetic Chemistry

Background:

• Fluorinases are enzymes enabling environmentally friendly fluorination.
• Natural fluorinase diversity is limited, necessitating protein engineering.
• Directed evolution offers a pathway to improve enzyme function.

Purpose of the Study:

• To engineer the fluorinase FlA1 for enhanced activity.
• To improve the conversion of 5 -chloro-5 -deoxyadenosine (5 -ClDA) to 5 -fluoro-5 -deoxyadenosine (5 -FDA).
• To apply evolved variants in the radiosynthesis of 5 -[18F]FDA.

Main Methods:

• Directed evolution of the fluorinase FlA1.
• Enzyme variant screening and characterization.
• Kinetic analysis of enzymatic reactions.
• Radiosynthesis using evolved fluorinase variants.

Main Results:

• Two evolved variants, fah2081 and fah2114, were identified.
• Variants demonstrated over 3-fold higher radiochemical conversion (RCC) for 5 -[18F]FDA radiosynthesis compared to wild-type FlA1.
• Kinetic studies revealed improved kcat for 5 -ClDA conversion but reduced kcat for SAM conversion in variants.

Conclusions:

• Directed evolution is effective for enhancing fluorinase activity.
• Engineered fluorinases show promise for improved radiosynthesis of 5 -FDA.
• Understanding kinetic alterations provides insights for further enzyme optimization.