Entropy drives selective fluorine recognition in PNAS PLUS the fluoroacetyl–CoA thioesterase from Streptomyces cattleya Amy M. Weeksa,1,2, Ningkun Wanga,1,3, Jeffrey G. Peltonb, and Michelle C. Y. Changa,c,4 aDepartment of Chemistry, University of California, Berkeley, CA 94720-1460; bQB3 Institute, University of California, Berkeley, CA 94720-1460; and cDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720-1460 Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved January 17, 2018 (received for review September 28, 2017) Fluorinated small molecules play an important role in the design of ognition of the fluorine substituent itself, the high polarization of bioactive compounds for a broad range of applications. As such, the C-F bond creates an opportunity for dipolar interactions to there is strong interest in developing a deeper understanding of occur with protein-based functional groups (14, 15), which appears how fluorine affects the interaction of these ligands with their to play a key role in the increased potency of ciprofloxacin (Cipro) targets. Given the small number of fluorinated metabolites iden- compared with analogs lacking the fluorine substituent (16, 17). tified to date, insights into fluorine recognition have been pro- Despite the strong interest in fluorine–protein interactions, vided almost entirely by synthetic systems. The fluoroacetyl–CoA there are few systems in which evolutionarily optimized inter- thioesterase (FlK) from Streptomyces cattleya thus provides a actions between ligand and receptor can be studied given the unique opportunity to study an enzyme–ligand pair that has been limited existence of naturally occurring fluorinated metabolites evolutionarily optimized for a surprisingly high 106 selectivity for a and macromolecules that interact with them. As one of the single fluorine substituent. In these studies, we synthesize a series few characterized organisms that produces fluorinated natural of analogs of fluoroacetyl–CoA and acetyl–CoA to generate non- products, Streptomyces cattleya provides an interesting system for hydrolyzable ester, amide, and ketone congeners of the thioester exploring native fluorine recognition by protein targets that have substrate to isolate the role of fluorine molecular recognition in been subject to an unusual evolutionary pressure to distinguish FlK selectivity. Using a combination of thermodynamic, kinetic, between fluorine and other similar substituents. One of the and protein NMR experiments, we show that fluorine recognition organofluorines produced by S. cattleya is fluoroacetate, a potent is entropically driven by the interaction of the fluorine substituent poison that relies on the inability of the target host’s primary with a key residue, Phe-36, on the lid structure that covers the metabolic enzymes to sufficiently discriminate against the con- active site, resulting in an ∼5- to 20-fold difference in binding servative replacement of hydrogen with fluorine (18, 19). S. cattleya K ( D). Although the magnitude of discrimination is similar to that itself produces a resistance protein, the fluoroacetyl CoA thi- found in designed synthetic ligand–protein complexes where di- oesterase (FlK) (20–22), which displays a remarkably high 106- polar interactions control fluorine recognition, these studies show fold preference for the fluorinated substrate compared with that hydrophobic and solvation effects serve as the major deter- minant of naturally evolved fluorine selectivity. Significance enzyme selectivity | fluorine | organofluorine | molecular recognition | Fluorination has become a key design strategy for the devel- metabolism opment of a broad range of small-molecule therapeutics. As a result, there is significant interest in understanding how fluo- ite-selective fluorination has become an important strategy in rine substituents affect interactions between ligands and their Sthe design of structurally diverse bioactive small molecules protein targets. In this context, we elucidate the molecular with a broad range of functions ranging from human therapeutics details of fluorine recognition in the fluoroacetyl–CoA thio- and imaging agents to biocides used for plants, fungi, and insects esterase, which has evolved a 106-fold selectivity for a single (1–4). The success of fluorine in the development of these fluorine atom as part of its native function in organofluorine compounds has mainly relied on the small size of fluorine to biosynthesis. Using a combination of thermodynamic, kinetic, conserve binding of the fluorinated ligand to its macromolecular and biophysical experiments, we show that fluorine recogni- target while tuning a breadth of pharmacological characteristics. tion is entropically driven by interaction with a hydrophobic As such, studies on the impact of fluorination on metabolic residue in the binding site. These findings highlight the im- stability, distribution, and other pharmacokinetic properties of portance of hydrophobic and solvation effects in mediating small molecules have provided important information on how fluorine-binding selectivity driven by natural selection. fluorine substituents contribute to their behavior in vivo (5, 6). In contrast, the role of fluorine in tuning binding affinity and se- Author contributions: A.M.W., N.W., J.G.P., and M.C.Y.C. designed research; A.M.W., lectivity of small molecules for their protein targets is relatively N.W., and J.G.P. performed research; A.M.W., N.W., J.G.P., and M.C.Y.C. analyzed data; less well understood given the exceptional and context-dependent and A.M.W., N.W., J.G.P., and M.C.Y.C. wrote the paper. elemental traits of fluorine (4). The authors declare no conflict of interest. ’ “ ” This article is a PNAS Direct Submission. Fluorine s high electronegativity and polar hydrophobicity, BIOCHEMISTRY as well as the highly polarized nature of the carbon–fluorine Published under the PNAS license. bond can contribute in different ways to protein–ligand inter- 1A.M.W. and N.W. contributed equally to this work. actions (4, 7). In some cases, nonspecific interactions arising 2Present address: Department of Pharmaceutical Chemistry, University of California, San from the increase in hydrophobic surface area introduced by Francisco, CA 94158. fluorine can exert an energetically significant effect on molecular 3Present address: Department of Chemistry, San Jose State University, San Jose, CA 95192. recognition (8–11). In others, the increased steric bulk of fluo- 4To whom correspondence should be addressed. Email: [email protected]. rine can cause changes in compound conformation that lead to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CHEMISTRY increased selectivity as observed in structure–activity relationship 1073/pnas.1717077115/-/DCSupplemental. studies on fluoxetine (Prozac) (12, 13). In terms of specific rec- Published online February 16, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1717077115 PNAS | vol. 115 | no. 10 | E2193–E2201 Downloaded by guest on September 29, 2021 acetyl–CoA, a cellularly abundant competitor (21). This selectivity 4 A B is largely derived from the increased kcat (10 -fold increase) due to 2.5 open FlK open closed change in the hydrolysis mechanism for the fluorinated substrate 2.0 apo FlK (23). Nevertheless, the remaining 100-fold difference attributed to lid 1.5 the decreased KM suggests that molecular recognition of the 1.0 fluorine substituent also plays a role in substrate discrimination 0.5 (21, 24). Given that the magnitude of the expected binding se- 0.0 lectivity is similar to that observed for synthetic organofluorine F36 -0.5 inhibitors (4, 14, 15), elucidation of the factors that control fluo- electron density (σ) -1.0 rine recognition in FlK can provide insight into optimizing the -1.5 design of fluorinated ligands. 0 50 100 150 200 250 300 350 Using a set of nonhydrolyzable substrate analogs to separate χ (degrees) the contributions of fluorine recognition from catalytic selectiv- ity, we show that FlK does indeed bind with increased affinity to Fig. 1. Conformational change observed in FlK upon soaking with substrate ligands containing a fluorine substituent (21). We further show or products. (A) Structural alignment of FlK in the presence and absence of that a mobile residue on a unique hydrophobic lid structure (21), products. Phe-36 is shown in blue for apo–FlK and in magenta for substrate- χ Phe-36, is involved in providing an entropic driving force for soaked FlK. (B) Ringer plot showing the Phe-36 -angle for FlK crystals in the fluorinated substrate binding and controlling the off-rate of the presence (open) and absence (closed) of substrate. Figures are based on PDB entry 3P2Q for apo FlK and 3P2S for open FlK. substrate. NMR studies were then carried out to examine FlK dynamics, which demonstrated that both fluorine and Phe-36 are required to lower the off-rate of the substrate. Taken together, well, with about 10% occupancy under cryocrystallography condi- these results support a model in which a conformationally dy- tions (Fig. 1B). namic residue (Phe-36) plays an important role in fluorine-based While biochemical characterization of the F36A mutant had substrate discrimination. In contrast to synthetic systems, in shown that kcat/KM for the fluoroacetyl–CoA was decreased by which enhanced ligand affinity is usually mediated by dipolar 140-fold without affecting acetyl–CoA parameters (21), the in- interactions that confer an enthalpic