Publications (9)36.73 Total impact
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Article: Activity of the Enterococcus faecalis EIIA(gnt) PTS component and its strong interaction with EIIB(gnt).
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ABSTRACT: Eubacteria can import and simultaneously phosphorylate a range of different carbohydrates by means of sugar specific phosphoenolpyruvate (PEP) dependent sugar phosphotransferase systems (PTSs). Here, we report the biochemical characterization of the gluconate specific PTS component EIIA(gnt) from Enterococcus faecalis and its unexpectedly strong complex with EIIB(gnt). We analyze the activity of the complex regarding phosphoryl transfer using kinetic measurements and demonstrate by mutagenesis that His-9 of EIIA(gnt) is essential for this process and represents most likely the phosphoryl group carrier of EIIA(gnt). With a combination of isothermal titration calorimetry (ITC), analytical ultracentrifugation (AUC), native gel electrophoresis and chemical crosslinking experiments we show that EIIA(gnt) and EIIB(gnt) form a strong 2:2 heterotetrameric complex, which seems to be destabilized upon phosphorylation of EIIB(gnt).Biochemical and Biophysical Research Communications 09/2009; 388(4):630-6. · 2.48 Impact Factor -
Article: Structure of the Enterococcus faecalis EIIA(gnt) PTS component.
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ABSTRACT: In Eubacteria, the utilization of a number of extracellular carbohydrates is mediated by sugar specific phosphoenolepyruvate (PEP) dependent sugar phosphotransferase systems (PTSs), which simultaneously import und phosphorylate their target sugars. Here, we report the crystal structure of the EIIA(gnt) component of the so far little investigated Enterococcus faecalis gluconate specific PTS. The crystal structure shows a tightly interacting dimer of EIIA(gnt) which is structurally similar to the related EIIA(man) from Escherichia coli. Homology modeling of E. faecalis HPr, EIIB(man) and their complexes with EIIA(man) suggests that despite moderate sequence identity between EIIA(man) and EIIA(gnt), the active sites closely match the situation observed in the E. coli system with His-9 of EIIA(gnt) being the likely phosphoryl group carrier. We therefore propose that the phosphoryl transfer reactions involving EIIA(gnt) proceed according to a mechanism analog to the one described for E. coli EIIA(man).Biochemical and Biophysical Research Communications 09/2009; 388(4):626-9. · 2.48 Impact Factor -
Article: Structure of the full-length enzyme I of the phosphoenolpyruvate-dependent sugar phosphotransferase system.
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ABSTRACT: Enzyme I (EI) is the phosphoenolpyruvate (PEP)-protein phosphotransferase at the entry point of the PEP-dependent sugar phosphotransferase system, which catalyzes carbohydrate uptake into bacterial cells. In the first step of this pathway EI phosphorylates the heat-stable phospho carrier protein at His-15 using PEP as a phosphoryl donor in a reaction that requires EI dimerization and autophosphorylation at His-190. The structure of the full-length protein from Staphylococcus carnosus at 2.5A reveals an extensive interaction surface between two molecules in adjacent asymmetric units. Structural comparison with related domains indicates that this surface represents the biochemically relevant contact area of dimeric EI. Each monomer has an extended configuration with the phosphohistidine and heat-stable phospho carrier protein-binding domains clearly separated from the C-terminal dimerization and PEP-binding region. The large distance of more than 35A between the active site His-190 and the PEP binding site suggests that large conformational changes must occur during the process of autophosphorylation, as has been proposed for the structurally related enzyme pyruvate phosphate dikinase. Our structure for the first time offers a framework to analyze a large amount of research in the context of the full-length model.Journal of Biological Chemistry 11/2006; 281(43):32508-15. · 4.77 Impact Factor -
Article: Solution structure of the active-centre mutant I14A of the histidine-containing phosphocarrier protein from Staphylococcus carnosus.
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ABSTRACT: High-pressure NMR experiments performed on the histidine-containing phosphocarrier protein (HPr) from Staphylococcus carnosus have shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds, 49 1H(N)-15N, and 25 1H(N)-1Halpha residual dipolar couplings, a well resolved three-dimensional structure could be determined. The overall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a displacement of the ring system of His15 and a distortion of the N-terminus of the first helix, which supports the histidine ring. In addition, the C-terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon, which is known to occupy hydrophobic cavities, causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phosphocarrier activity of the mutant and agree well with the earlier suggestion that Ile14 represents an anchoring point stabilizing the active-centre loop in its correct conformation.European Journal of Biochemistry 01/2005; 271(23-24):4815-24. · 3.58 Impact Factor -
Article: High-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus and characterization of its interaction with the bifunctional HPr kinase/phosphorylase.
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ABSTRACT: A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus was obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints. Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond. Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) of Staphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46. The dissociation constant Kd was determined to be 0.10 +/- 0.02 mM at 303 K from the NMR data, assuming independent binding. The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution. Using transversal relaxation optimized spectroscopy-heteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex. As expected, it covers the region around Ser46 and the small helix b following this residue. In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15. This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity. In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex from Lactobacillus casei shows interactions with the two phosphorylation sites. However, the NMR data also suggest differences for the full-length protein from S. xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein of S. xylosus. In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the complex of HPr with the core of HPrK/P of L. casei in crystals.Journal of Bacteriology 10/2004; 186(17):5906-18. · 3.83 Impact Factor -
Article: NMR-spectroscopic mapping of an engineered cavity in the I14A mutant of HPr from Staphylococcus carnosus using xenon.
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ABSTRACT: The interaction between the histidine-containing phosphocarrier protein HPr and xenon atoms in solution is studied in the present paper. Wild-type HPr as well as the exchange mutant I14A have been studied. Specific binding of xenon into an engineered cavity created via the exchange of amino acid residue I14 by alanine could be shown using 1H-15N heteronuclear single-quantum coherence (HSQC) spectroscopy. Xenon binding results in pronounced changes of the 1H and 15N chemical shifts of amide groups close to the cavity. In addition to this observation which allows the NMR-spectroscopic mapping of such cavities, we have shown that the entire molecule is slightly rearranged as a result of xenon binding. In contrast, wild-type HPr only exhibits minor chemical shift changes due to the nonspecific interactions with the xenon atoms in solution.Journal of the American Chemical Society 08/2003; 125(29):8726-7. · 9.91 Impact Factor -
Article: Structure of the full-length HPr kinase/phosphatase from Staphylococcus xylosus at 1.95 A resolution: Mimicking the product/substrate of the phospho transfer reactions.
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ABSTRACT: The histidine containing phospho carrier protein (HPr) kinase/phosphatase is involved in carbon catabolite repression, mainly in Gram-positive bacteria. It is a bifunctional enzyme that phosphorylates Ser-46-HPr in an ATP-dependent reaction and dephosphorylates P-Ser-46-HPr. X-ray analysis of the full-length crystalline enzyme from Staphylococcus xylosus at a resolution of 1.95 A shows the enzyme to consist of two clearly separated domains that are assembled in a hexameric structure resembling a three-bladed propeller. The N-terminal domain has a betaalphabeta fold similar to a segment from enzyme I of the sugar phosphotransferase system and to the uridyl-binding portion of MurF; it is structurally organized in three dimeric modules exposed to form the propeller blades. Two unexpected phosphate ions associated with highly conserved residues were found in the N-terminal dimeric interface. The C-terminal kinase domain is similar to that of the Lactobacillus casei enzyme and is assembled in six copies to form the compact central hub of the propeller. Beyond previously reported similarity with adenylate kinase, we suggest evolutionary relationship with phosphoenolpyruvate carboxykinase. In addition to a phosphate ion in the phosphate-binding loop of the kinase domain, we have identified a second phosphate-binding site that, by comparison with adenylate kinases, we believe accommodates a product/substrate phosphate, normally covalently linked to Ser-46 of HPr. Thus, we propose that our structure represents a product/substrate mimic of the kinase/phosphatase reaction.Proceedings of the National Academy of Sciences 04/2002; 99(6):3458-63. · 9.68 Impact Factor -
Article: Activity of the Enterococcus faecalis EIIAgnt PTS component and its strong interaction with EIIBgnt
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ABSTRACT: Eubacteria can import and simultaneously phosphorylate a range of different carbohydrates by means of sugar specific phosphoenolpyruvate (PEP) dependent sugar phosphotransferase systems (PTSs). Here, we report the biochemical characterization of the gluconate specific PTS component EIIAgnt from Enterococcus faecalis and its unexpectedly strong complex with EIIBgnt. We analyze the activity of the complex regarding phosphoryl transfer using kinetic measurements and demonstrate by mutagenesis that His-9 of EIIAgnt is essential for this process and represents most likely the phosphoryl group carrier of EIIAgnt. With a combination of isothermal titration calorimetry (ITC), analytical ultracentrifugation (AUC), native gel electrophoresis and chemical crosslinking experiments we show that EIIAgnt and EIIBgnt form a strong 2:2 heterotetrameric complex, which seems to be destabilized upon phosphorylation of EIIBgnt.Biochemical and Biophysical Research Communications. -
Article: Structure of the Enterococcus faecalis EIIAgnt PTS component
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ABSTRACT: In Eubacteria, the utilization of a number of extracellular carbohydrates is mediated by sugar specific phosphoenolepyruvate (PEP) dependent sugar phosphotransferase systems (PTSs), which simultaneously import und phosphorylate their target sugars. Here, we report the crystal structure of the EIIAgnt component of the so far little investigated Enterococcus faecalis gluconate specific PTS. The crystal structure shows a tightly interacting dimer of EIIAgnt which is structurally similar to the related EIIAman from Escherichia coli. Homology modeling of E. faecalis HPr, EIIBman and their complexes with EIIAman suggests that despite moderate sequence identity between EIIAman and EIIAgnt, the active sites closely match the situation observed in the E. coli system with His-9 of EIIAgnt being the likely phosphoryl group carrier. We therefore propose that the phosphoryl transfer reactions involving EIIAgnt proceed according to a mechanism analog to the one described for E. coli EIIAman.Biochemical and Biophysical Research Communications.
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Institutions
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2002–2009
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Ruhr-Universität Bochum
Bochum, North Rhine-Westphalia, Germany -
European Molecular Biology Laboratory
Heidelberg, Baden-Wuerttemberg, Germany
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