L Hederstedt

Lund University, Lund, Skane, Sweden

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Publications (111)433.09 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Most aerobic organisms contain catalase which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process.
    The Journal of biological chemistry. 08/2014;
  • Ewa Bukowska-Faniband, Lars Hederstedt
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    ABSTRACT: The nonessential process of peptidoglycan synthesis during Bacillus subtilis sporulation is one model to study bacterial cell wall biogenesis. SpoVD is a class B high-molecular weight penicillin-binding protein that is specific for sporulation. Strains lacking this protein produce spores without the peptidoglycan cortex layer and are heat-sensitive. The detailed functions of the four different protein domains of the SpoVD protein are unknown and the observed phenotype of strains lacking the entire protein could be an indirect defect. We therefore inactivated the transpeptidase domain by substitution of the active site serine residue. Our results demonstrate that endospore cortex synthesis depends on the transpeptidase activity of SpoVD specifically. This article is protected by copyright. All rights reserved.
    FEMS Microbiology Letters 06/2013; · 2.05 Impact Factor
  • Michael Baureder, Lars Hederstedt
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    ABSTRACT: Lactic acid bacteria (LAB) are of profound importance in food production and infection medicine. LAB do not rely on heme (protoheme IX) for growth and are unable to synthesize this cofactor but are generally able to assemble a small repertoire of heme-containing proteins if heme is provided from an exogenous source. These features are in contrast to other bacteria, which synthesize their heme or depend on heme for growth. We here present the cellular function of heme proteins so far identified in LAB and discuss their biogenesis as well as applications of the extraordinary heme physiology of LAB.
    Advances in Microbial Physiology 01/2013; 62C:1-43. · 6.55 Impact Factor
  • Michael Baureder, Ronny Reimann, Lars Hederstedt
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    ABSTRACT: Enterococcus faecalis exhibits high resistance to oxidative stress. Several enzymes are responsible for this trait. The role of alkyl hydroperoxide reductase (Ahp), thiol peroxidase (Tpx), and NADH peroxidase (Npr) in oxidative stress defense was recently characterized. Enterococcus faecalis, in contrast to many other streptococci, contains a catalase (KatA), but this enzyme can only be formed when the bacterium is supplied with heme. We have used this heme dependency of catalase activity and mutants deficient in KatA and Npr to investigate the role of the catalase in resistance against exogenous and endogenous hydrogen peroxide stress. The results demonstrate that in the presence of environmental heme catalase contributes to the protection against toxic effects of hydrogen peroxide.
    FEMS Microbiology Letters 04/2012; 331(2):160-4. · 2.05 Impact Factor
  • Lars Hederstedt
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    ABSTRACT: Respiration in plants, most animals and many aerobic microbes is dependent on heme A. This is a highly specialized type of heme found as prosthetic group in cytochrome a-containing respiratory oxidases. Heme A differs structurally from heme B (protoheme IX) by the presence of a hydroxyethylfarnesyl group instead of a vinyl side group at the C2 position and a formyl group instead of a methyl side group at position C8 of the porphyrin macrocycle. Heme A synthase catalyzes the formation of the formyl side group and is a poorly understood heme-containing membrane bound atypical monooxygenase. This review presents our current understanding of heme A synthesis at the molecular level in mitochondria and aerobic bacteria. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.
    Biochimica et Biophysica Acta 03/2012; 1817(6):920-7. · 4.66 Impact Factor
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    Michael Baureder, Lars Hederstedt
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    ABSTRACT: Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly.
    PLoS ONE 01/2012; 7(5):e36725. · 3.73 Impact Factor
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    Jörg Simon, Lars Hederstedt
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    ABSTRACT: Organisms employ one of several different enzyme systems to mature cytochromes c. The biosynthetic process involves the periplasmic reduction of cysteine residues in the heme c attachment motif of the apocytochrome, transmembrane transport of heme b and stereospecific covalent heme attachment via thioether bonds. The biogenesis System II (or Ccs system) is employed by β-, δ- and ε-proteobacteria, Gram-positive bacteria, Aquificales and cyanobacteria, as well as by algal and plant chloroplasts. System II comprises four (sometimes only three) membrane-bound proteins: CcsA (or ResC) and CcsB (ResB) are the components of the cytochrome c synthase, whereas CcdA and CcsX (ResA) function in the generation of a reduced heme c attachment motif. Some ε-proteobacteria contain CcsBA fusion proteins constituting single polypeptide cytochrome c synthases especially amenable for functional studies. This minireview highlights the recent findings on the structure, function and specificity of individual System II components and outlines the future challenges that remain to our understanding of the fascinating post-translational protein maturation process in more detail.
    FEBS Journal 09/2011; 278(22):4179-88. · 4.25 Impact Factor
  • Michael Baureder, Lars Hederstedt
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    ABSTRACT: Cytochrome b₅₅₈ of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b₅₅₈. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b₅₅₈ produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature.
    Protein Expression and Purification 05/2011; 80(1):97-101. · 1.43 Impact Factor
  • Myriam Brugna, Lena Tasse, Lars Hederstedt
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    ABSTRACT: Haem (protohaem IX) analogues are toxic compounds and have been considered for use as antibacterial agents, but the primary mechanism behind their toxicity has not been demonstrated. Using the haem protein catalase in the Gram-positive bacterium Enterococcus faecalis as an experimental system, we show that a variety of haem analogues can be taken up by bacterial cells and incorporated into haem-dependent enzymes. The resulting cofactor-substituted proteins are dysfunctional, generally resulting in arrested cell growth or death. This largely explains the cell toxicity of haem analogues. In contrast to many other organisms, E. faecalis does not depend on haem for growth, and therefore resists the toxicity of many haem analogues. We have exploited this feature to establish a bacterial in vivo system for the production of cofactor-substituted haem protein variants. As a pilot study, we produced, isolated and analysed novel catalase variants in which the iron atom of the haem prosthetic group is replaced by other metals, i.e. cobalt, gallium, tin, and zinc, and also variants containing meso-protoheme IX, ruthenium meso-protoporphyrin IX and (metal-free) protoporphyrin IX. Engineered haem proteins of this type are of potential use within basic research and the biotechnical industry.
    FEBS Journal 06/2010; 277(12):2663-72. · 4.25 Impact Factor
  • Myriam Brugna, Lena Tasse, Lars Hederstedt
    FEBS Journal 01/2010; · 4.25 Impact Factor
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    ABSTRACT: Six-coordinated heme groups are involved in a large variety of electron transfer reactions because of their ability to exist in both the ferrous (Fe2+) and ferric (Fe3+) state without any large differences in structure. Our studies on hemes coordinated by two histidines (bis-His) and hemes coordinated by histidine and methionine (His-Met) will be reviewed. In both of these coordination environments, the heme core can exhibit ferric low spin (electron paramagnetic resonance EPR) signals with large gmax values (also called Type I, highly anisotropic low spin, or highly axial low spin, HALS species) as well as rhombic EPR (Type II) signals. In bis-His coordinated hemes rhombic and HALS envelopes are related to the orientation of the His groups with respect to each other such that (i) parallel His planes results in a rhombic signal and (ii) perpendicular His planes results in a HALS signal. Correlation between the structure of the heme and its ligands for heme with His-Met axial ligation and ligand-field parameters, as derived from a large series of cytochrome c variants, show, however, that for such a combination of axial ligands there is no clear-cut difference between the large gmax and the “small g-anisotropy” cases as a result of the relative Met-His arrangements. Nonetheless, a new linear correlation links the average shift 〈δ〉 of the heme methyl groups with the gmax values. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1064–1082, 2009.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
    Biopolymers 11/2009; 91(12):1064 - 1082. · 2.88 Impact Factor
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    ABSTRACT: The bacterial spore, the hardiest known life form, can survive in a metabolically dormant state for many years and can withstand high temperatures, radiation, and toxic chemicals. The molecular basis of spore dormancy and resistance is not understood, but the physical state of water in the different spore compartments is thought to play a key role. To characterize this water in situ, we recorded the water (2)H and (17)O spin relaxation rates in D(2)O-exchanged Bacillus subtilis spores over a wide frequency range. The data indicate high water mobility throughout the spore, comparable with binary protein-water systems at similar hydration levels. Even in the dense core, the average water rotational correlation time is only 50 ps. Spore dormancy therefore cannot be explained by glass-like quenching of molecular diffusion but may be linked to dehydration-induced conformational changes in key enzymes. The data demonstrate that most spore proteins are rotationally immobilized, which may contribute to heat resistance by preventing heat-denatured proteins from aggregating irreversibly. We also find that the water permeability of the inner membrane is at least 2 orders of magnitude lower than for model membranes, consistent with the reported high degree of lipid immobilization in this membrane and with its proposed role in spore resistance to chemicals that damage DNA. The quantitative results reported here on water mobility and transport provide important clues about the mechanism of spore dormancy and resistance, with relevance to food preservation, disease prevention, and astrobiology.
    Proceedings of the National Academy of Sciences 11/2009; 106(46):19334-9. · 9.81 Impact Factor
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    ABSTRACT: We present a mass spectrometry-based method for the identification and quantification of membrane proteins using the low-specificity protease Proteinase K, at very high pH, to digest proteins isolated by a modified SDS-PAGE protocol. The resulting peptides are modified with a fragmentation-directing isotope labeled tag. We apply the method to quantify differences in membrane protein expression of Bacillus subtilis grown in the presence or absence of glucose.
    Journal of Proteome Research 11/2009; 8(12):5666-73. · 5.06 Impact Factor
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    ABSTRACT: The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulphide bonds in hitherto unknown proteins in the intermembrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high-molecular-weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae, confirmed that a disulphide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD, stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulphide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.
    Molecular Microbiology 11/2009; 75(1):46-60. · 4.96 Impact Factor
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    ABSTRACT: Cytochromes of the c-type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacter CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.
    Molecular Microbiology 09/2009; 73(6):1058-71. · 4.96 Impact Factor
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    ABSTRACT: BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca(2+), at an inter-domain position some 14 A away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca(2+)-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.
    Journal of Biological Chemistry 07/2009; 284(35):23719-33. · 4.65 Impact Factor
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    ABSTRACT: Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pK(a) values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.
    Journal of Biological Chemistry 02/2009; 284(15):10056-66. · 4.65 Impact Factor
  • Anna Lewin, Xiao-Dong Su, Lars Hederstedt
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    ABSTRACT: Plasmid pLALA was constructed for glycerol or glycerol-3-phosphate inducible plasmid-borne gene expression in Bacillus subtilis and closely related Gram-positive bacteria. Gene expression using pLALA is based on anti-termination of transcription and involves the B. subtilis GlpP protein that in the presence of glycerol-3-phosphate acts as an anti-terminator protein by binding to the 5'-untranslated region of glpD mRNA. Properties and the usefulness of the system, denoted LALA, were validated by inducible production in B. subtilis strains of two water-soluble proteins (beta-galactosidase and a protein phospho-tyrosine phosphatase), and one integral membrane protein (heme A synthase). Advantages with LALA is that it is based on positive control, does not involve a DNA-binding protein, and that glycerol, a cheap and stable compound, can be used as inducer of gene expression.
    Journal of Molecular Microbiology and Biotechnology 11/2008; 17(2):61-70. · 1.95 Impact Factor
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    Mirja Carlsson Möller, Lars Hederstedt
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    ABSTRACT: The trxA gene is regarded as essential in Bacillus subtilis, but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is expected if the TrxA protein is important for the activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phosphosulfate reductase. We demonstrate here that a TrxA deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD deficiency, indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.
    Journal of bacteriology 08/2008; 190(13):4660-5. · 3.94 Impact Factor
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    ABSTRACT: ResA is an extracytoplasmic membrane-bound thiol-disulfide oxidoreductase required for cytochrome c maturation in Bacillus subtilis. Previous biochemical and structural studies have revealed that the active-site cysteinyls cycle between oxidized and reduced states with a low reduction potential and that, upon reduction, a hydrophobic cavity forms close to the active site. Here we report in vivo studies of ResA-deficient B. subtilis complemented with a series of ResA variants. Using a range of methods to analyze the cellular cytochrome c content, we demonstrated (i) that the N-terminal transmembrane segment of ResA serves principally to anchor the protein to the cytoplasmic membrane but also plays a role in mediating the activity of the protein; (ii) that the active-site cysteines are important for cytochrome c maturation activity; (iii) that Pro141, which forms part of the hydrophobic cavity and which adopts a cis conformation, plays an important role in protein stability; (iv) that Glu80, which lies at the base of the hydrophobic cavity, is important for cytochrome c maturation activity; and, finally, (v) that Pro141 and Glu80 ResA mutant variants promote selective maturation of low levels of one c-type cytochrome, subunit II of the cytochrome c oxidase caa(3), indicating that this apocytochrome is distinct from the other three endogenous c-type cytochromes of B. subtilis.
    Journal of bacteriology 08/2008; 190(13):4697-705. · 3.94 Impact Factor

Publication Stats

2k Citations
433.09 Total Impact Points

Institutions

  • 1986–2013
    • Lund University
      • • Department of Biology
      • • Division of Applied Microbiology
      Lund, Skane, Sweden
  • 2011
    • Technical University Darmstadt
      • Research Area of Microbiology and Genetics
      Darmstadt, Hesse, Germany
  • 2009
    • ETH Zurich
      • Institute of Microbiology
      Zürich, ZH, Switzerland
  • 2008–2009
    • University of East Anglia
      • Centre for Molecular and Structural Biochemistry
      Norwich, ENG, United Kingdom
  • 1995
    • Lomonosov Moscow State University
      • A. N. Belozersky Research Institute of Physico-Chemical Biology
      Moscow, Moscow, Russia
  • 1992
    • Karolinska Institutet
      Solna, Stockholm, Sweden
  • 1989
    • University of California, Berkeley
      • Lawrence Berkeley Laboratory
      Berkeley, MO, United States