Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro
Abstract
The fluorinase enzyme represents the only biological mechanism capable of forming stable C–F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding – likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.
Citation
Kittilä , T , Calero , P , Fredslund , F , Lowe , P T , Tezé , D , Nieto-Domínguez , M , O’Hagan , D , Nikel , P I & Welner , D H 2022 , ' Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro ' , Microbial Biotechnology , vol. Early View . https://doi.org/10.1111/1751-7915.14009
Publication
Microbial Biotechnology
Status
Peer reviewed
ISSN
1751-7915Type
Journal article
Description
This work was funded by The Novo Nordisk Foundation grant to the Center for Biosustainability (NNF10CC1016517). P.I.N. was funded by grants from The Novo Nordisk Foundation (NNF20CC0035580, and LiFe, NNF18OC0034818), the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 814418 (SinFonia) and the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B). T.K. and M.N.D. were funded by fellowships from the European Union's Horizon 2020 research and innovation program under a Marie Skłodowska Curie project under grant agreement No. 713683 (COFUNDfellowsDTU).Collections
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