Directed evolution of a fluorinase for improved fluorination efficiency on a non-native substrate
September 24-28, 2017
Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered via directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of a non-native substrate, 5’-chloro-5’-deoxyadenosine (5’-ClDA), to 5’-fluoro-5’-deoxyadenosine (5’-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5’-[18F]FDA, with overall radiochemical conversion (RCC) >3-fold higher than FlA1. Kinetic studies of the 2-step reaction revealed that the variants significantly improved kcat in the conversion of 5’-ClDA to S-adenosyl-L-methionine (SAM) but decreased kcat in the conversion of SAM to 5’-FDA.
Huihua Sun, "Directed evolution of a fluorinase for improved fluorination efficiency on a non-native substrate" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/enzyme_xxiv/158