Gunhild Layer

Technische Universität Braunschweig, Brunswyck, Lower Saxony, Germany

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Publications (31)112.11 Total impact

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    ABSTRACT: Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1.
    No preview · Article · Sep 2014 · Journal of Biological Chemistry
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    ABSTRACT: The isobacteriochlorin heme d1 serves as an essential cofactor in the cytochrome cd1 nitrite reductase NirS which plays an important role for denitrification. During the biosynthesis of heme d1 the enzyme siroheme decarboxylase catalyzes the conversion of siroheme to 12,18-didecarboxysiroheme. This enzyme was discovered recently (Bali et al. (2011) Proc. Natl. Acad. Sci. USA 108, 18260-5) and is only scarcely characterized. Here, we present the crystal structure of the siroheme decarboxylase from Hydrogenobacter thermophilus representing the first three-dimensional structure for this type of enzyme. The overall structure strikingly resembles those of transcriptional regulators of the Lrp/AsnC-family. Moreover, the structure of the enzyme in complex with a substrate analog reveals first insights into its active site architecture. Through site-directed mutagenesis and subsequent biochemical characterization of the enzyme variants two conserved histidine residues within the active site are identified to be involved in substrate binding and catalysis. Based on our results we propose a potential catalytic mechanism for the enzymatic reaction catalyzed by the siroheme decarboxylase.
    No preview · Article · Jul 2014 · Journal of Molecular Biology
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    ABSTRACT: In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called "classical" heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793) was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458) was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.
    Full-text · Article · Jan 2014 · Archaea
  • A. -L. Kaufholz · G. Layer · D. Heinz · M. Jahn · D. Jahn

    No preview · Article · Nov 2013
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    Preview · Article · Jul 2013 · Biochemical Journal
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    ABSTRACT: The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors heme c and heme d1. Whereas the heme c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the heme d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential heme d1 insertion protein NirN in vivo. This NirS-NirN interaction is dependent on the presence of the putative heme d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipo-protein in P. aeruginosa. Finally, the analysis by UV-visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa wild type and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of heme d1 biosynthesis and insertion of the cofactor into NirS.
    Full-text · Article · May 2013 · Bioscience Reports
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    ABSTRACT: The first enzyme of heme biosynthesis, 5-aminolevulinic acid synthase, catalyses the pyridoxal-5'-phosphate-dependent condensation of glycine and succinyl-CoA to 5-aminolevulinic acid, CO2 and CoA. The crystal structure of Rhodobacter capsulatus ALAS provides first snapshots of the structural basis for substrate binding and catalysis. To elucidate the functional role of single amino acid residues in the active site for substrate discrimination, substrate positioning, catalysis and involved structural protein rearrangements, multiple mutant ALAS variants were generated. The quinonoid intermediates I and II were visualized in single turnover experiments, indicating the presence of an α-amino-β-ketoadipate intermediate. Further evidence was obtained by pH-dependent formation of quinonoid II formation from the product 5-aminolevulinic acid. The function of residues R21, T83, N85 and I86, all involved in coordination of the succinyl-CoA substrate carboxyl group, were analysed kinetically. Residues R21, T83 and I86, all provided by the second subunit to the inter subunit active site, were found essential. Their location on the second subunit provides the basis for the required structural dynamics during the complex condensation of both substrates. Utilization of L-alanine by the ALAS variant T83S indicated the importance of this residue for the discrimination of the glycine substrate against related amino acids. N85 was found solely important for succinyl-CoA substrate recognition and discrimination. Obtained results provide a novel dynamic view on the structural basis of ALAS substrate binding and catalysis.
    Full-text · Article · Jan 2013 · Biochemical Journal
  • Gunhild Layer · Martin J. Warren

    No preview · Chapter · Aug 2012
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    ABSTRACT: Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 μM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.
    Full-text · Article · Dec 2011 · Biochemical Journal
  • Sonja Storbeck · Gunhild Layer
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    ABSTRACT: Die Biosynthese von Häm d 1 in denitrifizierenden Bakterien und Häm in Archaea verläuft ungewübnlicherweise über die methylierte Vorstufe Precorrin-2. Die beteiligten Uroporphyrinogen-III-Methyltransferasen nutzen Arginin als katalytisch aktive Base. The biosynthesis of heme d 1 in denitrifying bacteria and heme in the Archaea proceeds via the methylated intermediate precorrin-2. The involved uroporphyrinogen III methyltransferases utilize arginine as catalytically active base.
    No preview · Article · Oct 2011 · BioSpektrum
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    ABSTRACT: During the biosynthesis of heme d1, the essential cofactor of cytochrome cd1 nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.
    Preview · Article · May 2011 · Journal of Biological Chemistry
  • Gunhild Layer · Dieter Jahn · Martina Jahn

    No preview · Chapter · Feb 2011
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    ABSTRACT: Heme is an essential prosthetic group for many proteins involved in fundamental biological processes in all three domains of life. In Eukaryota and Bacteria heme is formed via a conserved and well-studied biosynthetic pathway. Surprisingly, in Archaea heme biosynthesis proceeds via an alternative route which is poorly understood. In order to formulate a working hypothesis for this novel pathway, we searched 59 completely sequenced archaeal genomes for the presence of gene clusters consisting of established heme biosynthetic genes and colocalized conserved candidate genes. Within the majority of archaeal genomes it was possible to identify such heme biosynthesis gene clusters. From this analysis we have been able to identify several novel heme biosynthesis genes that are restricted to archaea. Intriguingly, several of the encoded proteins display similarity to enzymes involved in heme d(1) biosynthesis. To initiate an experimental verification of our proposals two Methanosarcina barkeri proteins predicted to catalyze the initial steps of archaeal heme biosynthesis were recombinantly produced, purified, and their predicted enzymatic functions verified.
    Full-text · Article · Dec 2010 · Archaea
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    ABSTRACT: Assembly of iron-sulfur (Fe-S) clusters and maturation of Fe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC2D complex, only in its reduced form (FADH2), which has the ability to reduce ferric iron. These results suggest that the SufBC2D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH2-dependent reductive mobilization of iron.
    No preview · Article · Jul 2010 · Journal of Biological Chemistry
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    Gunhild Layer · Joachim Reichelt · Dieter Jahn · Dirk W Heinz
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    ABSTRACT: Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V-shaped glutamyl-tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the "Radical SAM enzyme" coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure-function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.
    Full-text · Article · Jun 2010 · Protein Science
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    ABSTRACT: Assembly of iron-sulfur (Fe-S) clusters and maturation of Fe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC(2)D complex, only in its reduced form (FADH(2)), which has the ability to reduce ferric iron. These results suggest that the SufBC(2)D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH(2)-dependent reductive mobilization of iron.
    Preview · Article · May 2010 · Journal of Biological Chemistry

  • No preview · Article · Mar 2010
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    ABSTRACT: During heme biosynthesis the oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of the two propionate side chains on rings A and B of coproporphyrinogen III to the corresponding vinyl groups to yield protoporphyrinogen IX. Here, the sequence of the two decarboxylation steps during HemN catalysis was investigated. A reaction intermediate of HemN activity was isolated by HPLC analysis and identified as monovinyltripropionic acid porphyrin by mass spectrometry. This monovinylic reaction intermediate exhibited identical chromatographic behavior during HPLC analysis as harderoporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18-tetramethylporphyrin). Furthermore, HemN was able to utilize chemically synthesized harderoporphyrinogen as substrate and converted it to protoporphyrinogen IX. These results suggest that during HemN catalysis the propionate side chain of ring A of coproporphyrinogen III is decarboxylated prior to that of ring B.
    No preview · Article · Nov 2009 · Biological Chemistry
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    ABSTRACT: Biosynthesis of heme d(1), the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd(1), requires the methylation of the tetrapyrrole precursor uroporphyrinogen III at positions C-2 and C-7. We produced Pseudomonas aeruginosa NirE, a putative S-adenosyl-L-methionine (SAM)-dependent uroporphyrinogen III methyltransferase, as a recombinant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography. Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer. NirE was shown to be a SAM-dependent uroporphyrinogen III methyltransferase that catalyzes the conversion of uroporphyrinogen III into precorrin-2 in vivo and in vitro. A specific activity of 316.8 nmol of precorrin-2 h(-1) x mg(-1) of NirE was found for the conversion of uroporphyrinogen III to precorrin-2. At high enzyme concentrations NirE catalyzed an overmethylation of uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin. Substrate inhibition was observed at uroporphyrinogen III concentrations above 17 microM. The protein did bind SAM, although not with the same avidity as reported for other SAM-dependent uroporphyrinogen III methyltransferases involved in siroheme and cobalamin biosynthesis. A P. aeruginosa nirE transposon mutant was not complemented by native cobA encoding the SAM-dependent uroporphyrinogen III methyltransferase involved in cobalamin formation. However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special requirement of NirE for heme d(1) biosynthesis.
    Full-text · Article · Sep 2009 · FEBS Journal
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    ABSTRACT: Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.
    Preview · Article · Jun 2007 · Journal of Biological Chemistry