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Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non‐native Substrate
Author(s) -
Sun Huihua,
Yeo Wan Lin,
Lim Yee Hwee,
Chew Xinying,
Smith Derek John,
Xue Bo,
Chan Kok Ping,
Robinson Robert C.,
Robins Edward G.,
Zhao Huimin,
Ang Ee Lui
Publication year - 2016
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201606722
Subject(s) - deoxyadenosine , chemistry , directed evolution , substrate (aquarium) , combinatorial chemistry , stereochemistry , radiosynthesis , enzyme , biochemistry , biology , mutant , microbiology and biotechnology , ecology , in vivo , gene
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 through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non‐native substrate 5′‐chloro‐5′‐deoxyadenosine (5′‐ClDA) into 5′‐fluoro‐5′‐deoxyadenosine (5′‐FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5′‐[ 18 F]FDA, with overall radiochemical conversion (RCC) more than 3‐fold higher than wild‐type FlA1. Kinetic studies of the two‐step reaction revealed that the variants show a significantly improved k cat value in the conversion of 5′‐ClDA into S ‐adenosyl‐ l ‐methionine (SAM) but a reduced k cat value in the conversion of SAM into 5′‐FDA.