TY - JOUR
T1 - Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro
AU - Kittila, Tiia-Riikka
AU - Calero Valdayo, Patricia Maria
AU - Fredslund, Folmer
AU - Lowe, Phillip T
AU - Tezé, David
AU - Nieto Domínguez, Manuel José
AU - O'Hagan, David
AU - Nikel, Pablo Ivan
AU - Welner, Ditte Hededam
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
U2 - 10.1111/1751-7915.14009
DO - 10.1111/1751-7915.14009
M3 - Journal article
C2 - 35084776
VL - 15
SP - 1622
EP - 1632
JO - Microbial Biotechnology
JF - Microbial Biotechnology
SN - 1751-7907
IS - 5
ER -