Dataset Information

Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese.

ABSTRACT: H(2)O(2) is commonly generated in biological habitats by environmental chemistry and by cellular immune responses. H(2)O(2) penetrates cells, disrupts metabolism, and blocks growth; it therefore is of interest to identify the major cellular molecules that H(2)O(2) damages and the strategies by which cells protect themselves from it. We used a strain of Escherichia coli that lacks catalases and peroxidases to impose protracted low-grade H(2)O(2) stress. Physiological analysis indicated that the pentose-phosphate pathway, in particular, was poisoned by submicromolar intracellular H(2)O(2). Assays determined that ribulose-5-phosphate 3-epimerase (Rpe) was specifically inactivated. In vitro studies demonstrated that Rpe employs a ferrous iron atom as a solvent-exposed cofactor and that H(2)O(2) rapidly oxidizes this metal in a Fenton reaction. The oxidized iron is released immediately, causing a loss of activity. Most Rpe proteins could be reactivated by remetallation; however, a small fraction of proteins were irreversibly damaged by each oxidation cycle, and so repeated cycles of oxidation and remetallation progressively led to permanent inactivation of the entire Rpe pool. Manganese import and iron sequestration are key elements of the H(2)O(2) stress response, and we found that manganese can activate Rpe in vitro in place of iron, converting the enzyme to a form that is unaffected by H(2)O(2). Indeed, the provision of manganese to H(2)O(2)-stressed cells protected Rpe and enabled the pentose-phosphate pathway to retain function. These data indicate that mononuclear iron enzymes can be primary targets of H(2)O(2) stress and that cells adapt by shifting from iron- to manganese-centered metabolism.

SUBMITTER: Sobota JM

PROVIDER: S-EPMC3069151 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese.

Sobota Jason M JM Imlay James A JA

Proceedings of the National Academy of Sciences of the United States of America 20110314 13

H(2)O(2) is commonly generated in biological habitats by environmental chemistry and by cellular immune responses. H(2)O(2) penetrates cells, disrupts metabolism, and blocks growth; it therefore is of interest to identify the major cellular molecules that H(2)O(2) damages and the strategies by which cells protect themselves from it. We used a strain of Escherichia coli that lacks catalases and peroxidases to impose protracted low-grade H(2)O(2) stress. Physiological analysis indicated that the p ...[more]

PMID: 21402925

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Project description:Pentose phosphate pathway (PPP) plays a crucial role in the maintenance of NADPH/NADP+ homeostasis and provides protection against oxidative stress through detoxification of the reactive oxygen species. Ribulose-5-phosphate epimerase (RPE) participates in catalysis of the interconversion of ribulose-5-phosphate (Ru5P) to xylulose-5-phosphate (Xu5P) during PPP, however the structural attributes of this enzyme are still underexplored in many human pathogens including leishmanial parasites. The present study focuses upon cloning, purification and characterization of RPE of Leishmania donovani (LdRPE) using various biophysical and structural approaches. Sequence analysis has shown the presence of trypanosomatid-specific insertions at the N-terminus that are absent in humans and other eukaryotes. Gel filtration chromatography indicated recombinant LdRPE to exist as a dimer in the solution. Circular dichroism studies revealed a higher alpha helical content at physiological pH and temperature that comparatively varies with changing these parameters. Additionally, intrinsic fluorescence and quenching studies of LdRPE have depicted that tryptophan residues are mainly buried in the hydrophobic regions, and the recombinant enzyme is moderately tolerant to urea. Moreover, homology modeling was employed to generate the three-dimensional structure of LdRPE followed by molecular docking with the substrate, product, and substrate analogues. The modeled structure of LdRPE unravelled the presence of conserved active site residues as well as a single binding pocket for the substrate and product, while an in silico study suggested binding of substrate analogues into a similar pocket with more affinity than the substrate. Additionally, molecular dynamics simulation analysis has deciphered complexes of LdRPE with most of the ligands exhibiting more stability than its apo form and lesser fluctuations in active site residues in the presence of ligands. Altogether, our study presents structural insights into leishmanial RPE that could provide the basis for its implication to develop potent antileishmanials.

Project description:The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these structural findings.

Dataset Information

Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese.

Publications

Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese.

Similar Datasets

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