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- Title
Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate.
- Authors
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
- Abstract
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′-[18F]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 kcat value in the conversion of 5′-ClDA into S-adenosyl- l-methionine (SAM) but a reduced kcat value in the conversion of SAM into 5′-FDA.
- Subjects
ORGANOFLUORINE compounds; FLUORINATION; BIOCHEMICAL substrates; ADENOSYLMETHIONINE; DEOXYADENOSINE; NUCLEOPHILIC substitution reactions
- Publication
Angewandte Chemie, 2016, Vol 128, Issue 46, p14489
- ISSN
0044-8249
- Publication type
Article
- DOI
10.1002/ange.201606722