Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro

Tiia Kittilä, Patricia Calero, Folmer Fredslund, Phillip T. Lowe, David Tezé, Manuel Nieto-Domínguez, David O’Hagan, Pablo I. Nikel*, Ditte H. Welner*

*Corresponding author for this work

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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.
Original languageEnglish
Number of pages11
JournalMicrobial Biotechnology
VolumeEarly View
Early online date27 Jan 2022
DOIs
Publication statusE-pub ahead of print - 27 Jan 2022

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