The fluorinase is an enzyme that catalyses the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and L-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80-87 % identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X-ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.
In this study, we probed the inhibition of pig heart citrate synthase (E.C. 4.1.3.7) by synthesising seven analogues either designed to mimic the proposed enolate intermediate in this enzyme reaction or developed from historical inhibitors. The most potent inhibitor was fluorovinyl thioether 9 (Ki=4.3 μm), in which a fluorine replaces the oxygen atom of the enolate. A comparison of the potency of 9 with that of its non‐fluorinated vinyl thioether analogue 10 (Ki=68.3 μm) revealed a clear “fluorine effect” favouring 9 by an order of magnitude. The dethia analogues of 9 and 10 proved to be poor inhibitors. A methyl sulfoxide analogue was a moderate inhibitor (Ki=11.1 μm), thus suggesting hydrogen bonding interactions in the enolate site. Finally, E and Z propenoate thioether isomers were explored as conformationally constrained carboxylates, but these were not inhibitors. All compounds were prepared by the synthesis of the appropriate pantetheinyl diol and then assembly of the coenzyme A structure according to a three‐enzyme biotransformation protocol. A quantum mechanical study, modelling both inhibitors 9 and 10 into the active site indicated short CF⋅⋅⋅H contacts of ≈2.0 Å, consistent with fluorine making two stabilising hydrogen bonds, and mimicking an enolate rather than an enol intermediate. Computation also indicated that binding of 9 to citrate synthase increases the basicity of a key aspartic acid carboxylate, which becomes protonated.
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