Molecular Recognition of Fluorine Impacts Substrate Selectivity in the Fluoroacetyl-CoA Thioesterase FlK

Abstract

The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH–rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and (S)-[2H1]fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-2H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme–substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts.

Natural production of fluorinated compounds and biotechnological prospects of the fluorinase enzyme

“…This reaction is very interesting not only because of its selectivity (the enzyme is 10 6 more selective for fluoroacetyl-CoA than for acetyl-CoA), but also because it may explain how S. cattleya resists the biochemical effects of FA. The catalytic mechanism and kinetics of this enzyme were studied in detail by Dias et al [138] and Weeks et al [139][140][141] The in vitro production of 4-FT through the overexpression and recombination of all the enzymes involved in the biosynthesis of this secondary fluorometabolite was achieved by Deng et al [142] in a work that not only proved the metabolic steps leading to the bioproduction of this amino acid but also demonstrated the importance that the fluorinase may have in the organofluorine production industry.…”

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

“…This selectivity is largely derived from the increased k cat (10 4 -fold increase) due to change in the hydrolysis mechanism for the fluorinated substrate (23). Nevertheless, the remaining 100-fold difference attributed to the decreased K M suggests that molecular recognition of the fluorine substituent also plays a role in substrate discrimination (21,24). Given that the magnitude of the expected binding selectivity is similar to that observed for synthetic organofluorine inhibitors (4,14,15), elucidation of the factors that control fluorine recognition in FlK can provide insight into optimizing the design of fluorinated ligands.…”

Synthetic Biology Approaches to Fluorinated Polyketides

“…22 For both substrates, a common covalent acyl-enzyme intermediate is generated on Glu50, with a rate constant that depends on the inductive polarization of the substrate carbonyl. 24 However, the next step diverges depending on the substrate. The acetyl acyl-enzyme intermediate undergoes slow hydrolysis by attack of water on the carbonyl moiety through a canonical thioesterase mechanism.…”