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

Abstract

Summary 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 ( K M ) 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.

A Nonconventional Archaeal Fluorinase Identified by In Silico Mining for Enhanced Fluorine Biocatalysis

“…In the cell-free extract assay, the final 5′-FDA concentrations detected were within the ranges previously reported. 27,38 Interestingly, no other fluorometabolites than 5′-FDA could be detected in these assays.…”

“…The alterations could be mapped near to the adenyl moiety of SAM, and involve the substitution of a conserved proline for an arginine residue and an RNAA motif for YYGG. This motif is found in the C -terminal domain of other fluorinases, which is more variable than the N -terminal domain and is presumably also involved in hexamer formation 38 ( Figure S4 ). Interestingly, the catalytic features found in FlA PtaU1 do not resemble those of the SalL Stro chlorinase, which would place FlA PtaU1 in a different functional group of S N 2 halogenases.…”

Identification of Two Novel Fluorinases From Amycolatopsis sp. CA-128772 and Methanosaeta sp. PtaU1.Bin055 and a Mutant With Improved Catalytic Efficiency With Native Substrate

“…Different from the reported enzymes, Fme also has the function of fluorinase, although no extra motif is present in the protein sequence. A recent study proves that the hexameric form of fluorinase is not necessary for catalytic activity, which explains the rationality of Fme activity ( Kittilä et al, 2022 ). According to previous reports, fluorinase can catalyze the chlorination reaction, while chlorinase cannot produce organic fluoride by using F ions.…”

A Non-conventional Archaeal Fluorinase Identified by In Silico Mining Catalyzes the Fastest Natural Nucleophilic Fluorination

“…This motif is found in the C -terminal domain of fluorinases, which is more variable than the N -terminal domain ( Fig. S4 ) and is presumably also involved in hexamer formation (Kittilä et al ., 2022). Interestingly, the variations found in FlA PtaU1 do not resemble those seen in the SalL Stro chlorinase.…”