Entropy drives selective fluorine recognition in the fluoroacetyl–CoA thioesterase from Streptomyces cattleya

Abstract

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl-CoA thioesterase (FlK) from thus provides a unique opportunity to study an enzyme-ligand pair that has been evolutionarily optimized for a surprisingly high 10 selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl-CoA and acetyl-CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding (). Although the magnitude of discrimination is similar to that found in designed synthetic ligand-protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity.

Defluorination of 4-fluorothreonine by threonine deaminase

“…S1 †). The reaction was monitored by 19 F-NMR analysis. However, there was no apparent new organofluorine signal appearing in the 19 F-NMR spectrum while 4-FT 2 from the FTaseMA biochemical reaction accumulated, suggesting that the fluorine atom at the C4 position of 4-FT 2 strongly inhibits the reactivity of LAAO.…”

“…1B). [17][18][19] The detoxification mechanism of 4-FT 2 employed by S. cattleya has also been extensively elaborated by the study of FthB and FthC, the fluorothreonyl-tRNA deacylase and 4-FT transporter, respectively. The structural similarity between 4-FT 2 and L-Thr allows 2 to form a misacylated tRNA, 4-FT-tRNA, hence entering the ribosomal protein biosynthesis.…”

“…Here we report the biochemical defluorination reaction of 4-FT 2, catalysed by threonine deaminase (TDA), which may serve an alternative pathway of 4-FT 2 to prevent its intracellular accumulation. The products, resulting from this defluorination reaction, were determined to be inorganic fluoride and 4-hydroxy-α-ketobutyrate through 19 F-NMR analysis, chemical derivatization and high-resolution mass spectroscopic analysis, and the latter product was proposed to be further degraded into pyruvate by the promiscuous enzyme 2-keto-4-hydroxyglutarate aldolase through comparative genomic analysis.…”

Enzymatic synthesis of fluorinated compounds

“…To understand the mechanism of selective fluorination, a comprehensive strategy was implemented for FLK including thermodynamic, kinetic, and biophysical experiments. These studies indicated that entropy drive mediated by hydrophobicity and solvation is the key to selective fluorination recognition, especially the substrate-binding controlled by the active site Phe36 (Figure S4 ) (Weeks et al 2018 ). Moreover, the selectivity to the fluoroacetyl-CoA substrate not only depends on the polarization provided by the negatively charged fluorine substitution but also on the molecular recognition of fluorine during the formation of acyl-FLK intermediate (Weeks et al 2014 ).…”

Halogenases for biosynthetic pathway engineering: Toward new routes to naturals and non-naturals

“…The fluoroacetyl-CoA hydrolase (FlK) and a trans acting fluorothreonyl-tRNA deacylase (FthB) is part of this enzymatic machinery in S. cattleya. [209,210,211] FlK enzyme hydrolyze fluoroacetyl-CoA, whereas FthB removes the misacylation and incorporation of fluorothreonyl-tRNA instead of L-threonine into proteins during the synthesis.…”