Structure of the bis divalent cation complex with phosphonoacetohydroxamate at the active site of enolase

Poyner, Russell R.; Reed, George H.

doi:10.1021/bi00146a020

Cited by 49 publications

(79 citation statements)

References 42 publications

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“…The presence of spinexchange coupling would provide clear evidence that the two Mn(II) ions are forming a binuclear cluster in AntR, as suggested from the MntR structure ( Figure 7). This approach has been used to identify the formation of a binuclear Mn-(II) cluster in aminopeptidases (30,31), concanavalin A (32), phosphatase (33), thiosulfate oxidase (34), arginase (35), phosphotriesterase (36), and enolase (37). As shown in Figure 4, our low-temperature cw EPR spectra lack the spectral signatures associated with spin-exchange coupling and therefore are not consistent with formation of a binuclear manganese cluster in AntR.…”

Section: Discussionmentioning

confidence: 90%

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

et al. 2006

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The anthracis repressor (AntR) is a manganese-activated transcriptional regulator from Bacillus anthracis and is a member of the diphtheria toxin repressor (DtxR) family of proteins. In this paper, we characterize the Mn(II) binding and protein dimerization state using a combination of continuous wave (cw) and pulsed EPR methods. Equilibrium metal binding experiments showed that AntR binds 2 equivalents of Mn(II) with positive cooperativity and apparent dissociation constants of 210 and 16.6 µM. AntR showed sub-millisecond Mn(II) on-rates as measured using stopped-flow EPR. The kinetics of Mn(II) dissociation, measured by displacement with Zn(II), was biphasic with rate constants of 35.7 and 0.115 s -1 . Variable-temperature parallel and perpendicular mode cw EPR spectra showed no evidence of a spin-exchange interaction, suggesting that the two Mn(II) ions are not forming a binuclear cluster. Finally, size exclusion chromatography and double electron-electron resonance EPR demonstrated that AntR forms a dimer in the absence of Mn(II). These results provide insights into the metal activation of AntR and allow a comparison with related DtxR proteins.Bacteria, like all organisms, regulate the amount of transition metal ions in the cell. Of these metals, iron is particularly important as it is essential for viability, but accumulation of iron, especially in the ferrous [Fe(II)] form, results in oxidative damage as the metal is oxidized to the ferric form. Bacteria generally do not accumulate iron or other transition metals as seen in eukaryotic cells and, therefore, require efficient mechanisms for regulating the intracellular concentration of transition metal ions (1). Iron homeostasis is regulated in many bacteria by proteins of the diphtheria toxin repressor (DtxR) 1 family. These proteins function as metal ion sensors and regulate the transcription of genes involved in metal uptake, storage, and utilization.DtxR, the prototypical member of this family of repressors, regulates more than 40 genes in Corynebacterium diphtheriae, and IdeR, from Mycobacterium tuberculosis, regulates approximately 45 different genes (2). In many cases, these repressors have been co-opted to regulate the virulence response (1, 3). In C. diphtheriae, toxin gene expression is repressed by the metal-activated form of DtxR when the intracellular Fe(II) levels are above a certain threshold. A drop in the Fe(II) level inactivates the repressor and allows active gene transcription.DtxR proteins typically contain two domains connected by a flexible tether (Figure 1).

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Section: Discussionmentioning

confidence: 90%

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

et al. 2006

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“…Whereas MR and MLE both require a divalent metal ion for activity, enolase requires two metal ions, bound to a high-affinity and a low-affinity site (23)(24)(25). The metal ligands in MR, MLE, and the high-affinity enolase site are quite similar (Fig.…”

Section: Resultsmentioning

confidence: 98%

Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

Hasson

Schlichting

Moulai

et al. 1998

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

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Muconate lactonizing enzyme (MLE), a component of the ␤-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the ''enolase superfamily'') that catalyze the abstraction of the ␣-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-Å resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn 2؉ -bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.

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“…The most potent enolase inhibitor described in the literature is Phosphoacetohydroxamate (PhAH, Fig. 1a), which has nM IC 50 inhibitory activity against Enolase from diverse sources 6,7 . Although several structures of human ENO2 have been reported 8 , PhAH-bound structures are only available for yeast and trypanosome Enolase 9-11 .…”

Section: Introductionmentioning

confidence: 99%

SF2312 is a natural phosphonate inhibitor of enolase

et al. 2016

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Despite being critical for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme Enolase 2 for the treatment of cancers with deletion of Enolase 1, we modeled the synthetic tool compound inhibitor, Phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analogue of PhAH, with the hydroxamic nitrogen linked to the alpha-carbon by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure based search revealed that our hypothesized back-bone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low nM inhibitor of Enolase.

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Structure of the bis divalent cation complex with phosphonoacetohydroxamate at the active site of enolase

Cited by 49 publications

References 42 publications

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

SF2312 is a natural phosphonate inhibitor of enolase

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