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

Huihua Sun; Wan Lin Yeo; Yee Hwee Lim; Xinying Chew; Derek John Smith; Bo Xue; Kok Ping Chan; Robert C Robinson; Edward G Robins; Huimin Zhao; Ee Lui Ang

doi:10.1002/anie.201606722

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

Angew Chem Int Ed Engl. 2016 Nov 7;55(46):14277-14280. doi: 10.1002/anie.201606722. Epub 2016 Oct 14.

Authors

Huihua Sun¹, Wan Lin Yeo¹, Yee Hwee Lim², Xinying Chew², Derek John Smith^{3

4}, Bo Xue⁵, Kok Ping Chan², Robert C Robinson^{5

6

7

8

9}, Edward G Robins¹⁰, Huimin Zhao^{11

12}, Ee Lui Ang¹³

Affiliations

¹ Metabolic Engineering Research Laboratory (MERL), Science and Engineering Institutes, Agency for Science, Technology, and Research (A*STAR), 31 Biopolis Way, Nanos #01-01, Singapore, 138669, Singapore.
² Institute of Chemical and Engineering Sciences (ICES), A*STAR, 8 Biomedical Grove, Neuros #07-01/02/03, Singapore, 138665, Singapore.
³ Bioinformatics Institute, A*STAR, 30 Biopolis Street, Matrix #07-01, Singapore, 138671, Singapore.
⁴ Biotransformation Innovation Platform, 61 Biopolis Drive, Proteos #04-14, Singapore, 138673, Singapore.
⁵ Institute of Molecular and Cell Biology (IMCB), A*STAR, 61 Biopolis Drive, Proteos #03-15, Singapore, 138673, Singapore.
⁶ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore.
⁷ NTU Institute of Structural Biology, Nanyang Technological University (NTU), 59 Nanyang Drive, Singapore, 636921, Singapore.
⁸ School of Biological Sciences, NTU, 60 Nanyang Drive, Singapore, 637551, Singapore.
⁹ Lee Kong Chian School of Medicine, 50 Nanyang Avenue, Singapore, 639798, Singapore.
¹⁰ Singapore Bioimaging Consortium (SBIC), A*STAR, 11 Biopolis way, #02-02, Singapore, 138667, Singapore.
¹¹ Metabolic Engineering Research Laboratory (MERL), Science and Engineering Institutes, Agency for Science, Technology, and Research (A*STAR), 31 Biopolis Way, Nanos #01-01, Singapore, 138669, Singapore. zhao5@illinois.edu.
¹² 215 Roger Adams Laboratory, Box C3, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA. zhao5@illinois.edu.
¹³ Metabolic Engineering Research Laboratory (MERL), Science and Engineering Institutes, Agency for Science, Technology, and Research (A*STAR), 31 Biopolis Way, Nanos #01-01, Singapore, 138669, Singapore. angel@merl.a-star.edu.sg.

PMID: 27739177
DOI: 10.1002/anie.201606722

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'-[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.

Keywords: biocatalysis; directed evolution; fluorinases; halogenation; radiosynthesis.

Publication types

Research Support, Non-U.S. Gov't