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Dieter Jahn - One of the best experts on this subject based on the ideXlab platform.

  • Leishmania major possesses a unique HemG-type protoporphyrinogen IX Oxidase
    Portland Press Biochemical Society, 2014
    Co-Authors: Dagmar Zwerschke, Dieter Jahn, Simone Karrie, Martina Jahn
    Abstract:

    Leishmania major was proposed to either utilize haem from its host or partially synthesize the tetrapyrrole from host provided precursors. However, only indirect evidence was available for this partial late haem biosynthetic pathway. Here, we demonstrate that the LMJF_06_1280 gene of L. major encodes a HemG-type PPO (protoporphyrinogen IX Oxidase) catalysing the oxidation of protoporphyrinogen IX to protoporphyrin IX. Interestingly, trypanosomatids are currently the only known eukaryotes possessing HemG-type enzymes. The LMJF_06_1280 gene forms a potential transcriptional unit with LMJF_06_1270 encoding CPO (Coproporphyrinogen III Oxidase) and with LMJF_06_1290 for a cytochrome b5. In vivo function of the L. major hemG gene was shown by the functional complementation of the Escherichia coli ΔhemG strain LG285. Restored haem formation in E. coli was observed using HPLC analyses. Purified recombinant L. major HemG revealed PPO activity in vitro using different ubiquinones and triphenyltetrazolium as electron acceptors. FMN was identified as the L. major HemG cofactor. Active site residues were found to be essential for HemG catalysis. These data in combination with the solved crystal structures of L. major CPO and the physiological proof of a ferrochelatase activity provide clear-cut evidence for a partial haem biosynthetic pathway in L. major

  • metabolic engineering of cobalamin vitamin b12 production in bacillus megaterium
    Microbial Biotechnology, 2010
    Co-Authors: Rebekka Biedendieck, Helen K Leech, Evelyne Deery, Marco Malten, Heiko Barg, Boyke Bunk, Janhenning Martens, Martin J Warren, Dieter Jahn
    Abstract:

    Cobalamin (vitamin B(12)) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B(12) biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG(A)cbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding Coproporphyrinogen III Oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B(12) riboswitch upstream of the cbiXJCDETLFGAcysG(A)cbiYbtuR operon and the recombinant production of three different vitamin B(12) binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B(12)-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B(12) concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.

  • complex formation between protoporphyrinogen ix Oxidase and ferrochelatase during haem biosynthesis in thermosynechococcus elongatus
    Microbiology, 2008
    Co-Authors: Ava Masoumi, Ilka U Heinemann, Martina Jahn, Michael Koch, Manfred Rohde, Dieter Jahn
    Abstract:

    During haem and chlorophyll biosynthesis, flavin-dependent protoporphyrinogen IX Oxidase catalyses the six-electron oxidation of protoporphyrinogen IX to form protoporphyrin IX. In the following step, iron is inserted into protoporphyrin IX by ferrochelatase. Based on the solved crystal structures of these enzymes, an in silico model for a complex between these two enzymes was proposed to protect the highly photoreactive intermediate protoporphyrin IX. The existence of this complex was verified by two independent techniques. First, co-immunoprecipitation experiments using antibodies directed against recombinantly produced and purified Thermosynechococcus elongatus protoporphyrinogen IX Oxidase and ferrochelatase demonstrated their physical interaction. Secondly, protein complex formation was visualized by in vivo immunogold labelling and electron microscopy with T. elongatus cells. Finally, oxygen-dependent Coproporphyrinogen III Oxidase, which catalyses the formation of protoporphyrinogen IX, was not found to be part of this complex when analysed with the same methodology.

  • radical s adenosylmethionine enzyme Coproporphyrinogen III Oxidase hemn functional features of the 4fe 4s cluster and the two bound s adenosyl l methionines
    Journal of Biological Chemistry, 2005
    Co-Authors: Gunhild Layer, Ava Masoumi, Martina Jahn, Katrin Grage, Daniela Breckau, Peter Heathcote, Thomas Teschner, Volker Schunemann, A X Trautwein, Dieter Jahn
    Abstract:

    Abstract The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent Coproporphyrinogen III Oxidase HemN catalyzes the oxidative decarboxylation of Coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis. The recently solved crystal structure of Escherichia coli HemN revealed the presence of an unusually coordinated iron-sulfur cluster and two molecules of AdoMet. EPR spectroscopy of the reduced iron-sulfur center in anaerobically purified HemN in the absence of AdoMet has revealed a [4Fe-4S]1+ cluster in two slightly different conformations. Mossbauer spectroscopy of anaerobically purified HemN has identified a predominantly [4Fe-4S]2+ cluster in which only three iron atoms were coordinated by cysteine residues (isomer shift of δ = 0.43 (1) mm/s). The fourth non-cysteine-ligated iron exhibited a δ = 0.57 (3) mm/s, which shifted to a δ = 0.68 (3) mm/s upon addition of AdoMet. Substrate binding by HemN did not alter AdoMet coordination to the cluster. Multiple rounds of AdoMet cleavage with the formation of the reaction product methionine indicated AdoMet consumption during catalysis and identified AdoMet as a co-substrate for HemN catalysis. AdoMet cleavage was found to be dependent on the presence of the substrate Coproporphyrinogen III. Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX. Finally, the binding site for the unusual second, non iron-sulfur cluster coordinating AdoMet molecule (AdoMet2) was targeted using site-directed mutagenesis. All AdoMet2 binding site mutants still contained an iron-sulfur cluster and most still exhibited AdoMet cleavage, albeit reduced compared with the wild-type enzyme. However, all mutants lost their overall catalytic ability indicating a functional role for AdoMet2 in HemN catalysis. The reported significant correlation of structural and functional biophysical and biochemical data identifies HemN as a useful model system for the elucidation of general AdoMet radical enzyme features.

  • crystal structure of Coproporphyrinogen III Oxidase reveals cofactor geometry of radical sam enzymes
    The EMBO Journal, 2003
    Co-Authors: Gunhild Layer, Jürgen Moser, Dirk W Heinz, Dieter Jahn, Wolfdieter Schubert
    Abstract:

    ‘Radical SAM’ enzymes generate catalytic radicals by combining a 4Fe–4S cluster and S ‐adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen‐independent Coproporphyrinogen III Oxidase, at 2.07 A resolution. HemN catalyzes the essential conversion of Coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe–4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 A) and a neighboring sulfur of the cluster (3.6 A), allowing single electron transfer from the 4Fe–4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5′‐deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.

Gunhild Layer - One of the best experts on this subject based on the ideXlab platform.

  • the oxygen independent Coproporphyrinogen III Oxidase hemn utilizes harderoporphyrinogen as a reaction intermediate during conversion of Coproporphyrinogen III to protoporphyrinogen ix
    Biological Chemistry, 2010
    Co-Authors: Katrin Rand, Dirk W Heinz, Claudia Noll, Hans Martin Schiebel, Dorit Kemken, Thomas Dulcks, Markus Kalesse, Gunhild Layer
    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.

  • the substrate radical of escherichia coli oxygen independent Coproporphyrinogen III Oxidase hemn
    Journal of Biological Chemistry, 2006
    Co-Authors: Gunhild Layer, Antonio J Pierik, Matthias Trost, Stephen E J Rigby, Helen K Leech, Katrin Grage, Daniela Breckau, I Astner, Lothar Jansch, Peter Heathcote
    Abstract:

    During porphyrin biosynthesis the oxygen-independent Coproporphyrinogen III Oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of Coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate Coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) Coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with Coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this Coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.

  • radical s adenosylmethionine enzyme Coproporphyrinogen III Oxidase hemn functional features of the 4fe 4s cluster and the two bound s adenosyl l methionines
    Journal of Biological Chemistry, 2005
    Co-Authors: Gunhild Layer, Ava Masoumi, Martina Jahn, Katrin Grage, Daniela Breckau, Peter Heathcote, Thomas Teschner, Volker Schunemann, A X Trautwein, Dieter Jahn
    Abstract:

    Abstract The S-adenosylmethionine (AdoMet) radical enzyme oxygen-independent Coproporphyrinogen III Oxidase HemN catalyzes the oxidative decarboxylation of Coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis. The recently solved crystal structure of Escherichia coli HemN revealed the presence of an unusually coordinated iron-sulfur cluster and two molecules of AdoMet. EPR spectroscopy of the reduced iron-sulfur center in anaerobically purified HemN in the absence of AdoMet has revealed a [4Fe-4S]1+ cluster in two slightly different conformations. Mossbauer spectroscopy of anaerobically purified HemN has identified a predominantly [4Fe-4S]2+ cluster in which only three iron atoms were coordinated by cysteine residues (isomer shift of δ = 0.43 (1) mm/s). The fourth non-cysteine-ligated iron exhibited a δ = 0.57 (3) mm/s, which shifted to a δ = 0.68 (3) mm/s upon addition of AdoMet. Substrate binding by HemN did not alter AdoMet coordination to the cluster. Multiple rounds of AdoMet cleavage with the formation of the reaction product methionine indicated AdoMet consumption during catalysis and identified AdoMet as a co-substrate for HemN catalysis. AdoMet cleavage was found to be dependent on the presence of the substrate Coproporphyrinogen III. Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX. Finally, the binding site for the unusual second, non iron-sulfur cluster coordinating AdoMet molecule (AdoMet2) was targeted using site-directed mutagenesis. All AdoMet2 binding site mutants still contained an iron-sulfur cluster and most still exhibited AdoMet cleavage, albeit reduced compared with the wild-type enzyme. However, all mutants lost their overall catalytic ability indicating a functional role for AdoMet2 in HemN catalysis. The reported significant correlation of structural and functional biophysical and biochemical data identifies HemN as a useful model system for the elucidation of general AdoMet radical enzyme features.

  • crystal structure of Coproporphyrinogen III Oxidase reveals cofactor geometry of radical sam enzymes
    The EMBO Journal, 2003
    Co-Authors: Gunhild Layer, Jürgen Moser, Dirk W Heinz, Dieter Jahn, Wolfdieter Schubert
    Abstract:

    ‘Radical SAM’ enzymes generate catalytic radicals by combining a 4Fe–4S cluster and S ‐adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen‐independent Coproporphyrinogen III Oxidase, at 2.07 A resolution. HemN catalyzes the essential conversion of Coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe–4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 A) and a neighboring sulfur of the cluster (3.6 A), allowing single electron transfer from the 4Fe–4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5′‐deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.

  • oxygen dependent Coproporphyrinogen III Oxidase hemf from escherichia coli is stimulated by manganese
    Journal of Biological Chemistry, 2003
    Co-Authors: Daniela Breckau, Gunhild Layer, Esther Mahlitz, Anselm Sauerwald, Dieter Jahn
    Abstract:

    Abstract During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of Coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent Coproporphyrinogen III Oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 μm for Coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored Coproporphyrinogen III Oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.

C N Hunter - One of the best experts on this subject based on the ideXlab platform.

Dingzhong Tang - One of the best experts on this subject based on the ideXlab platform.

Baiming Cui - One of the best experts on this subject based on the ideXlab platform.

  • a mutation in a Coproporphyrinogen III Oxidase gene confers growth inhibition enhanced powdery mildew resistance and powdery mildew induced cell death in arabidopsis
    Plant Cell Reports, 2013
    Co-Authors: Chuanyu Guo, Jin Xing, Dingzhong Tang, Baiming Cui
    Abstract:

    A gene encoding a Coproporphyrinogen III Oxidase mediates disease resistance in plants by the salicylic acid pathway. A number of genes that regulate powdery mildew resistance have been identified in Arabidopsis, such as ENHANCED DISEASE RESISTANCE 1 to 3 (EDR1 to 3). To further study the molecular interactions between the powdery mildew pathogen and Arabidopsis, we isolated and characterized a mutant that exhibited enhanced resistance to powdery mildew. The mutant also showed dramatic powdery mildew-induced cell death as well as growth defects and early senescence in the absence of pathogens. We identified the affected gene by map-based cloning and found that the gene encodes a Coproporphyrinogen III Oxidase, a key enzyme in the tetrapyrrole biosynthesis pathway, previously known as LESION INITIATION 2 (LIN2). Therefore, we designated the mutant lin2-2. Further studies revealed that the lin2-2 mutant also displayed enhanced resistance to Hyaloperonospora arabidopsidis (H.a.) Noco2. Genetic analysis showed that the lin2-2-mediated disease resistance and spontaneous cell death were dependent on PHYTOALEXIN DEFICIENT 4 (PAD4), SALICYLIC ACID INDUCTION-DEFICIENT 2 (SID2), and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), which are all involved in salicylic acid signaling. Furthermore, the relative expression levels of defense-related genes were induced after powdery mildew infection in the lin2-2 mutant. These data indicated that LIN2 plays an important role in cell death control and defense responses in plants.