Bernd Hoffmann

University of Vienna, Wien, Vienna, Austria

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Publications (8)22.78 Total impact

  • Journal of Biomolecular NMR 08/2007; 38(3):267. · 2.85 Impact Factor
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    ABSTRACT: Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K(D)) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel beta-barrel with the strand order (-beta1 -beta2 -beta3 -beta6 -beta5 -beta4 -beta7 -beta8), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.
    Journal of Bacteriology 02/2007; 189(1):254-64. · 3.19 Impact Factor
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    ABSTRACT: In structural proteomics, it is necessary to efficiently screen in a high-throughput manner for the presence of stable structures in proteins that can be subjected to subsequent structure determination by X-ray or NMR spectroscopy. Here we illustrate that the (1)H chemical distribution in a protein as detected by (1)H NMR spectroscopy can be used to probe protein structural stability (e.g., the presence of stable protein structures) of proteins in solution. Based on experimental data obtained on well-structured proteins and proteins that exist in a molten globule state or a partially folded alpha-helical state, a well-defined threshold exists that can be used as a quantitative benchmark for protein structural stability (e.g., foldedness) in solution. Additionally, in this chapter we describe a largely automated strategy for rapid fold validation and structure-based backbone signal assignment. Our methodology is based on a limited number of NMR experiments (e.g., HNCA and 3D NOESY-HSQC) and performs a Monte Carlo-type optimization. The novel feature of the method is the opportunity to screen for structural fragments (e.g., template scanning). The performance of this new validation tool is demonstrated with applications to a diverse set of proteins.
    Methods in Enzymology 02/2005; 394:142-75. · 2.00 Impact Factor
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    Journal of Biomolecular NMR 12/2004; 30(3):361-2. · 2.85 Impact Factor
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    ABSTRACT: Pseudocoenzyme B12 (=Coβ-(5′-deoxy-5′-adenosyl)-(adenin-7-yl)cobamide; 1) and adenosyl-factor A (=Coβ-(5′-deoxy-5′-adenosyl)-(2-methyladenin-7-yl)cobamide; 3) are two natural analogues of coenzyme B12 (=adenosylcobalamin-Coβ-(5′-deoxy-5′-adenosyl)-(5,6-dimethyl-1H-benzimidazolyl)cobamide; 2), where the Co-coordinating 5,6-dimethyl-1H-benzimidazole nucleotide base of 2 is replaced by the purine bases adenine and 2-methyladenine. In contrast to 2, which exists solely in the ‘base-on' form, UV/VIS spectroscopy qualitatively indicates ‘base-off' constitution for 1 and 3 in aqueous solution. (cf. the established ‘base-off' form as unexpected binding mode of B12 cofactors in several B12-dependent enzymes, such as in methionine synthase from Escherichia coli and in glutamate mutase from Clostridium cochlearium). In the present work, pseudocoenzyme B12 (1) was synthesized in 85% yield by alkylation with 5′-O-tosyladenosine of (adenin-7-yl)cob(I)amide, which was produced electrochemically from pseudovitamin B12 (Coβ-cyano-(adenin-7-yl)cobamide). Likewise, adenosyl-factor A (3) was prepared in ca. 70% yield from factor A (=Coβ-cyano-(2-methyladenin-7-yl)cobamide; 5). All the spectroscopic properties of 1 and 3 in aqueous solution indicated that these two Coβ-(5′-deoxy-5′-adenosyl)-(adenin-7-yl)cobamides exist predominantly in a ‘base-off' constitution, with minor but significant contributions of the ‘base-on' form. From the UV/VIS spectra, the temperature-dependent equilibrium constants of the ‘base-off'/‘base-on' reconstitution reaction were determined as Kon (1)=0.30 and Kon (3)=0.48 at 25°, corresponding to a contribution of the ‘base-on' forms of 23% for 1 and of 32% for 3.
    Helvetica Chimica Acta 01/2002; 85:927-944. · 1.38 Impact Factor
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    ABSTRACT: Uniformly (13)C,(15)N-labeled MutS, the coenzyme B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum, was prepared by overexpression from an Escherichia coli strain. Multidimensional heteronuclear NMR spectroscopic experiments with aqueous solutions of (13)C,(15)N-labeled MutS provided signal assignments for roughly 90% of the 1025 hydrogen, 651 carbon, and 173 nitrogen atoms and resulted in about 1800 experimental restraints. Based on the information from the NMR experiments, the structure of MutS was calculated, confirming the earlier, less detailed structure obtained with (15)N-labeled MutS. The refined analysis allowed a precise determination of the secondary and tertiary structure including several crucial side chain interactions. The structures of (the apoprotein) MutS in solution and of the B(12)-binding subunit in the crystal of the corresponding homologous holoenzyme from Clostridium cochlearium differ only in a section that forms the well-structured helix alpha1 in the crystal structure and that also comprises the cobalt-coordinating histidine residue. In the apoprotein MutS, this part of the B(12)-binding subunit is dynamic. The carboxy-terminal end of this section is conformationally flexible and has significant propensity for an alpha-helical structure ("nascent helix"). This dynamic section in MutS is a decisive element for the binding of the nucleotide moiety of coenzyme B(12) and appears to be stabilized as a helix (alpha1) upon trapping of the nucleotide of the B(12) cofactor.
    ChemBioChem 10/2001; 2(9):643-55. · 3.74 Impact Factor
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    ABSTRACT: The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Co(beta)-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 micromol) of B(12) derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample of C. cochlearium by a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B(12) (Co(beta)-cyano-7"-adeninylcobamide) (60%) and factor A (Co(beta)-cyano-7"-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B(12) was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B(12) and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional (1)H, (13)C-, and (15)N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-[N(6)-methyl]-adeninylcobamide, a previously unknown corrinoid. C. cochlearium thus biosynthesizes as its native "complete" B(12) cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.
    Journal of Bacteriology 10/2000; 182(17):4773-82. · 3.19 Impact Factor
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    ABSTRACT: Glutamate mutase (Glm) is an adenosylcobamide-dependent enzyme that catalyzes the reversible rearrangement of (2S)-glutamate to (2S, 3S)-3-methylaspartate. The active enzyme from Clostridium cochlearium consists of two subunits (of 53.6 and 14.8 kDa) as an alpha2beta2 tetramer, whose assembly is mediated by coenzyme B12. The smaller of the protein components, GlmS, has been suggested to be the B12-binding subunit. Here we report the solution structure of GlmS, determined from a heteronuclear NMR-study, and the analysis of important dynamical aspects of this apoenzyme subunit. The global fold and dynamic behavior of GlmS in solution are similar to those of the corresponding subunit MutS from C. tetanomorphum, which has previously been investigated using NMR-spectroscopy. Both solution structures of the two Glm B12-binding subunits share striking similarities with that determined by crystallography for the B12-binding domain of methylmalonyl CoA mutase (Mcm) from Propionibacterium shermanii, which is B12 bound. In the crystal structure a conserved histidine residue was found to be coordinated to cobalt, displacing the endogenous axial ligand of the cobamide. However, in GlmS and MutS the sequence motif, Asp-x-His-x-x-Gly, which includes the cobalt-coordinating histidine residue, and a predicted alpha-helical region following the motif, are present as an unstructured and highly mobile loop. In the absence of coenzyme, the B12-binding site apparently is only partially formed. By comparing the crystal structure of Mcm with the solution structures of B12-free GlmS and MutS, a consistent picture on the mechanism of B12 binding has been obtained. Important elements of the binding site only become structured upon binding B12; these include the cobalt-coordinating histidine residue, and an alpha helix that forms one side of the cleft accommodating the nucleotide 'tail' of the coenzyme.
    European Journal of Biochemistry 08/1999; 263(1):178-88. · 3.58 Impact Factor

Publication Stats

65 Citations
22.78 Total Impact Points

Institutions

  • 2005–2007
    • University of Vienna
      • Institut für Organische Chemie
      Wien, Vienna, Austria
  • 1999–2002
    • University of Innsbruck
      • Institute of Organic Chemistry
      Innsbruck, Tyrol, Austria
  • 2000
    • Universität Ulm
      • Institute of Microbiology and Biotechnology
      Ulm, Baden-Württemberg, Germany