title: Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate.
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 kcatvalue in the conversion of 5'-ClDA into S-adenosyl-l-methionine (SAM) but a reduced kcatvalue in the conversion of SAM into 5'-FDA.
authors.affiliation: 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.
authors.forename: Huihua
authors.initials: H
authors.lastname: Sun
authors.affiliation: 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.
authors.forename: Wan Lin
authors.initials: WL
authors.lastname: Yeo
authors.affiliation: Institute of Chemical and Engineering Sciences (ICES), A*STAR, 8 Biomedical Grove, Neuros #07-01/02/03, Singapore, 138665, Singapore.
authors.forename: Yee Hwee
authors.initials: YH
authors.lastname: Lim
authors.affiliation: Institute of Chemical and Engineering Sciences (ICES), A*STAR, 8 Biomedical Grove, Neuros #07-01/02/03, Singapore, 138665, Singapore.
authors.forename: Xinying
authors.initials: X
authors.lastname: Chew
authors.affiliation: Bioinformatics Institute, A*STAR, 30 Biopolis Street, Matrix #07-01, Singapore, 138671, Singapore.|Biotransformation Innovation Platform, 61 Biopolis Drive, Proteos #04-14, Singapore, 138673, Singapore.
authors.forename: Derek John
authors.initials: DJ
authors.lastname: Smith
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR, 61 Biopolis Drive, Proteos #03-15, Singapore, 138673, Singapore.
authors.forename: Bo
authors.initials: B
authors.lastname: Xue
authors.affiliation: Institute of Chemical and Engineering Sciences (ICES), A*STAR, 8 Biomedical Grove, Neuros #07-01/02/03, Singapore, 138665, Singapore.
authors.forename: Kok Ping
authors.initials: KP
authors.lastname: Chan
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR, 61 Biopolis Drive, Proteos #03-15, Singapore, 138673, Singapore.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597, Singapore.|NTU Institute of Structural Biology, Nanyang Technological University (NTU), 59 Nanyang Drive, Singapore, 636921, Singapore.|School of Biological Sciences, NTU, 60 Nanyang Drive, Singapore, 637551, Singapore.|Lee Kong Chian School of Medicine, 50 Nanyang Avenue, Singapore, 639798, Singapore.
authors.forename: Robert C
authors.identifier: 0000-0001-6367-6903
authors.initials: RC
authors.lastname: Robinson
authors.affiliation: Singapore Bioimaging Consortium (SBIC), A*STAR, 11 Biopolis way, #02-02, Singapore, 138667, Singapore.
authors.forename: Edward G
authors.initials: EG
authors.lastname: Robins
authors.affiliation: 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.|215 Roger Adams Laboratory, Box C3, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA. zhao5@illinois.edu.
authors.forename: Huimin
authors.identifier: 0000-0002-9069-6739
authors.initials: H
authors.lastname: Zhao
authors.affiliation: 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.
authors.forename: Ee Lui
authors.identifier: 0000-0002-3070-8056
authors.initials: EL
authors.lastname: Ang
keywords: biocatalysis; directed evolution; fluorinases; halogenation; radiosynthesis