Fluorocitrate Inhibition of Aconitase: Relative Configuration of Inhibitory Isomer by X-ray Crystallography

Carrell, H. L.; Glusker, Jenny P.; Villafranca, Joseph J.; Mildvan, Albert S.; Dummel, Robert J.; Kun, Ernest

doi:10.1126/science.170.3965.1412

Cited by 63 publications

(28 citation statements)

References 12 publications

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“…Interestingly, many mutants in this class are involved in acetate metabolism, including TCA cycle genes, suggesting flux of bromoacetate through metabolism as a mechanism of detoxification. This is not without precedent as fluoroacetate enters the acetate metabolic pathway, and its toxicity is due to the lethal synthesis of the aconitase inhibitor, fluorocitrate (28). Also prevalent in this class are genes involved in synthesizing and reducing ubiquinone and genes in the cysteine biosynthetic pathway.…”

Section: Resultsmentioning

confidence: 99%

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

Desai

Miller

2010

Proc. Natl. Acad. Sci. U.S.A.

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Microbial niches contain toxic chemicals capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival in the presence of a normally lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP- N -acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. We also screened a comprehensive library of E. coli single-gene deletion mutants and identified 63 strains displaying increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ , a gene encoding a predicted glutathione transferase. Herein, YliJ is shown to catalyze the glutathione-dependent dehalogenation of bromoacetate with a k cat / K m value of 5.4 × 10 3 M -1 s -1 . YliJ displays exceptional substrate specificity and produces a rate enhancement exceeding 5 orders of magnitude, remarkable characteristics for reactivity with a nonnatural molecule. This study illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins.

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Section: Resultsmentioning

confidence: 99%

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

Desai

Miller

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…The synthesis and purification of the four different diastereoisomers of 2-fluorocitrate by Kun and Dummel (9) led to the identification of (Ϫ)-erythro-2-fluorocitrate, (2R,3R), as the inhibitory isomer of the enzyme (10). Kinetic studies of this compound with aconitase have yielded a range of K I values and have demonstrated that depending on which substrate was used in the studies, inhibition may be either competitive or noncompetitive and that this competition is reversible (11)(12)(13).…”

mentioning

confidence: 99%

The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex

Lauble

Kennedy

Emptage

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

114

121

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It has been known for many years that f luoroacetate and f luorocitrate when metabolized are highly toxic, and that at least one effect of f luorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (؊)-erythro diastereomer of 2-f luorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to f luoro-cis-aconitate, followed by addition of hydroxide and with loss of f luoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. Formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of f luorocitrate inhibition is supported by the crystal structure of the enzymeinhibitor complex at 2.05-Å resolution, release of f luoride stoichiometric with total enzyme when (؊)-erythro-2-f luorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 10 6 -fold excess of isocitrate, and previously published Mössbauer experiments. When (؉)-erythro-2-f luorocitrate is added to aconitase, the release of f luoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative ␣-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (؊)-erythro-2-f luorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural substrate, isocitrate. The structure exhibits four hydrogen bonds <2.7 Å in length involving HTn, H 2 O bound to the [4Fe-4S] cluster, Asp-165 and His-167, as well as low temperature factors for these moieties, consistent with the observed very tight binding of the inhibitor.The mechanism of the inhibitory effects of fluorocitrate on the enzyme aconitase [citrate(isocitrate)hydrolyase, EC 4.2.1.3] has been a long-standing problem in biochemistry. The toxic nature of fluoroacetate was discovered over 50 years ago (1, 2) and citrate was found to accumulate in tissues poisoned with compounds that could provide the fluoroacetyl residue. On this basis Peters (3) and Martius (4) proposed that the inhibitory substance was a fluorotricarboxylic acid. Subsequently it was shown that 2-fluorocitrate is indeed produced metabolically via the citrate synthase reaction (5) and that in the presence of this substance the enzyme aconitase is inhibited (6). Aconitase catalyzes the conversion of citrate to isocitrate (Iso) via the obligatory intermediate cis-aconitate (Scheme I).

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“…By comparison with synthetic standards, the enzymic product was one of the erythro pair rather than the threo pair, in fact, the erythro isomer with (-) rotation (44). An X-ray structure of the mixture of epimeric kerythro 2-fluorocitrate salts established that (-) erythro had either 2R,3R, or 2S,3S absolute configuration (45). Here the matter rested, although Kun hypothesized correctly the 2R,3R structure based on anticipated regiospecificity of citrate synthase in putting an acetate (F-acetate) unit into the pro S arm of citrate (44).…”

Section: Fluorinated Substrate Analogsmentioning

confidence: 94%

Fluorinated Substrate Analogs: Routes of Metabolism and Selective Toxicity

Walsh

1983

Advances in Enzymology - And Related Areas of Molecular Biology

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Fluorocitrate Inhibition of Aconitase: Relative Configuration of Inhibitory Isomer by X-ray Crystallography

Cited by 63 publications

References 12 publications

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex

Fluorinated Substrate Analogs: Routes of Metabolism and Selective Toxicity

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