Catalytic control of enzymatic fluorine specificity Amy M. Weeksa and Michelle C. Y. Changa,b,c,1 Departments of aChemistry and bMolecular and Cell Biology, University of California, Berkeley, CA 94720; and cPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Edited by Perry Allen Frey, University of Wisconsin-Madison, Madison, WI, and approved October 24, 2012 (received for review July 21, 2012) The investigation of unique chemical phenotypes has led to the The potency of fluoroacetate as a poison poses an especially discovery of enzymes with interesting behaviors that allow us to difficult substrate selectivity problem at the cellular level for explore unusual function. The organofluorine-producing microbe the host organism, where a single fluorine atom must be rec- Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase ognized very specifically over hydrogen to clear very low en- (FlK) that demonstrates a surprisingly high level of discrimination dogenous levels of toxic fluoroacetyl-CoA while simultaneously for a single fluorine substituent on its substrate compared with maintaining the high levels of acetyl-CoA (5)requiredforcell the cellularly abundant hydrogen analog, acetyl-CoA. In this re- growth and survival. Consequently, there exists a natural se- port, we show that the high selectivity of FlK is achieved through lective pressure on S. cattleya to meet this challenge, and it has catalysis rather than molecular recognition, where deprotonation evolved a fluoroacetyl-CoA thioesterase (FlK) that catalyzes fl at the Cα position to form a putative ketene intermediate only the breakdown of uoroacetyl-CoA to prevent lethal synthesis occurs on the fluorinated substrate, thereby accelerating the rate of fluorocitrate (Fig. 1A)(22–24).Wehaverecentlyshownthat 6 of hydrolysis 104-fold compared with the nonfluorinated conge- FlK demonstrates an extremely high, 10 -fold selectivity for fl ner. These studies provide insight into mechanisms of catalytic hydrolysis of uoroacetyl-CoA over acetyl-CoA based on a de- K 2 k 4 fl selectivity in a native system where the existence of two reaction crease in m (10 ) and increase in cat (10 )forthe uorinated K fl pathways determines substrate rather than product selection. substrate (24). Although the m of FlK with respect to uo- roacetyl-CoA (8 μM) is significantly lower than that for acetyl- enzyme mechanism | substrate selectivity CoA (2 mM), it is interesting to note that FlK likely operates near saturation for both substrates in vivo, given the high in- tracellular concentration of acetyl-CoA (25), with substrate iving organisms have solved some of the most difficult selectivity governed by the rate of hydrolysis (kcat)ratherthan Lchallenges in catalysis by harnessing the exquisite selectivity K substrate binding ( d). We thus set out to explore the molec- and reactivity of enzyme active sites to build complex chemical ular origin of the catalytic selectivity of FlK with regard to behaviors (1–5). Indeed, the plasticity of enzymes toward fl fl uorine and now report the existence of an unexpected Cα- evolution of new function has allowed life to ourishindiverse deprotonation pathway for enzymatic thioester hydrolysis, biospheres by conferring a selective advantage to hosts that through a putative ketene intermediate, that serves as the basis can demonstrate catalytic innovation and use unusual resour- for fluorine discrimination. ces. The enzymes that form the molecular basis for these chemical phenotypes are a rich source of molecular diversity Results and provide an experimental platform for the continual search to gain insight into the mechanisms and dynamics of protein Elucidating the Kinetic Basis for Fluorine Selectivity. The hydrolysis and organismal evolution (6–9). One of the salient features of of thioesters typically involves direct attack of the nucleophile at enzymes is their extremely high substrate selectivity, which the carbonyl group to form a tetrahedral intermediate, which subsequently collapses to produce the free carboxylic acid and allows them to choose the correct substrate over the thousands fl of other small molecules that are present in cells at any given thiol end products. The uorine substitution itself activates the time. Thus, the exploration of substrate specificity and its carbonyl moiety toward nucleophilic attack through inductive evolution is key for understanding both enzyme catalysis and the effects and provides a potential mechanism for discrimination – between fluoroacetyl- and acetyl-CoA (SI Appendix, Fig. S1). expansion of biodiversity at the molecular level (7 12). fl In this context, a singular chemical trait found in the soil However, the rate of chemical hydrolysis of uoroacetyl-CoA at Streptomyces cattleya neutral pH is only 10-fold faster compared with acetyl-CoA, bacterium is its ability to catalyze the for- 4 mation of carbon–fluorine bonds (13, 14). Fluorine resides at rather than 10 -fold, as occurs in the FlK active site (24). Even at pH >13, the largest difference in reactivity that we observe for one corner of the periodic table, and its unique elemental SI Appendix properties make it highly effective as a design element for drug chemical hydrolysis is 100-fold ( , Fig. S2). These fl observations suggest that the large rate acceleration of FlK with discovery but also quite challenging for the synthesis of uorine- fl containing molecules (15–17). Although fluorine has emerged as respect to uoroacetyl-CoA may originate from more than a BIOCHEMISTRY a common motif in synthetic compounds, including top-selling simple increase in the electrophilicity of the carbonyl group upon fl drugs such as atorvastatin and fluticasone, it has been under- uorine substitution. To further probe the role of carbonyl ac- exploited in nature, and only a few biogenic organofluorine tivation in FlK substrate selectivity, we prepared a set of acyl-CoA 3 5–11 α compounds (<20) have been identified to date despite the analogs ( , ) with different functional groups at the position, thousands of known natural products containing chlorine and thereby accessing a wide range of values of the polar substituent σ bromine (∼5,000) (18, 19). In fact, the only fully characterized constant ( *) that describes the activation of the acyl-CoA car- organofluorine natural products are derived from a single path- bonyl group toward nucleophilic attack (26). By examining the σ way that produces the deceptively simple poison fluoroacetate relationship between * and the rate of reaction, this series of (1). The high toxicity of fluoroacetate arises from its antime- tabolite mode of action resulting from the substitution of one hydrogen in acetate (2), an important cellular building block, Author contributions: A.M.W. and M.C.Y.C. designed research; A.M.W. performed re- with fluorine. As a consequence of this conservative structural search; A.M.W. and M.C.Y.C. analyzed data; and A.M.W. and M.C.Y.C. wrote the paper. change, fluoroacetate can still enter normal acetate metabolism The authors declare no conflict of interest. via activation to fluoroacetyl-CoA (3) but then acts as a mecha- This article is a PNAS Direct Submission. nism-based inhibitor of the tricarboxylic acid (TCA) cycle upon 1To whom correspondence should be addressed. E-mail: [email protected]. fl 4 fl conversion to uorocitrate ( ) and elimination of uoride at This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a critical point (20, 21) (Fig. 1A). 1073/pnas.1212591109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1212591109 PNAS | November 27, 2012 | vol. 109 | no. 48 | 19667–19672 Downloaded by guest on September 28, 2021 _ Biosynthesis COO A R = H _ _ TCA cycle OOC COO Energy production R = H OH Biosynthesis _ Cell growth Acs or AckA/Pta Citrate synthase OOC OH Aconitase Isocitrate O O _ _ OOC COO R _ R O FlK SCoA R _ COO 4 _ _ R = F (1, 3) Antibiotic resistance OOC COO Aconitase inhibition H (2, 5) Growth defects R = F TCA cycle shutdown OH Cytotoxicity 106 Selectivity for R = F 4-Hydroxytransaconitate k B O H2O ( uncat) O C O Acylation O O Deacylation O R or R _ + CoA R R R _ k2 k3 O SCoA FlK (kcat) O SCoA O Glu 50 CoA Acyl-enzyme intermediate Uncatalyzed hydrolysis FlK-catalyzed hydrolysis Acetyl-CoA (R = H) Fluoroacetyl-CoA (R = F) -3.2 3.0 60 450 k k F 2(acylation) > 3(deacylation) -3.4 2.5 400 CN 2.0 50 -3.6 Cl 350 -3.8 F 1.5 Cl M) M) μ 40 μ 1.0 CN 300 -4.0 Br cat uncat 0.5 Br 250 -4.2 Ac 30 0.0 Ac 200 -4.4 Log k Log k -0.5 20 150 -4.6 -1.0 Me 100 -4.8 Me -1.5 H CoA formed ( 10 CoA formed ( H 50 -5.0 Et -2.0 Et -5.2 -2.5 0 0 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0246810 0 5 10 15 20 σ* σ* Time (s) Time (s) Fig. 1. FlK selectivity for fluoroacetyl-CoA over acetyl-CoA is derived from a change in the rate-limiting step in thioester hydrolysis. (A) FlK catalyzes the hydrolysis of fluoroacetyl-CoA, which reverses the lethal synthesis of an irreversible aconitase inhibitor. (B) Taft free-energy relationship analysis of unca- talyzed (Left) and FlK-catalyzed (Right) acyl-CoA hydrolysis. Pseudo-first-order rate constants were determined by linear fitting (for uncatalyzed reactions) or by fitting a plot of initial rate versus substrate concentration to the Michaelis-Menten equation (for FlK-catalyzed reactions). Ac, acetyl; Et, ethyl; Me, methyl. (C) Pre-steady-state kinetics of FlK-catalyzed acetyl-CoA (Left,25μM FlK) and fluoroacetyl-CoA hydrolysis (Right; red squares, 25 μM FlK; black squares, 50 μM FlK; grey squares, 75 μM FlK).