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Marjorie A Jones - One of the best experts on this subject based on the ideXlab platform.

  • normal and abnormal heme biosynthesis 6 synthesis and metabolism of a series of monovinylporphyrinogens related to harderoporphyrinogen further insights into the oxidative decarboxylation of porphyrinogen substrates by Coproporphyrinogen oxidase
    Journal of Organic Chemistry, 2010
    Co-Authors: Timothy D Lash, Ukti N Mani, Annasigrid I M Keck, Marjorie A Jones
    Abstract:

    A series of vinylporphyrinogens were prepared to probe the enzyme Coproporphyrinogen oxidase (CPO). Six (2-chloroethyl)porphyrins were synthesized from a common dipyrrylmethane via a,c-biladiene intermediates in excellent yields. Subsequent dehydrohalogenation with DBU in refluxing DMF then gave the required vinylporphyrin methyl esters, including harderoporphyrin-I, harderoporphyrin-III, and isoharderoporphyrin. The corresponding porphyrinogen carboxylic acids were incubated with chicken red cell hemolysates, which contain the enzyme CPO, and the products analyzed. The 17-ethyl analogue of harderoporphyrinogen-III, but not its 13-ethyl isomer, was shown to be an excellent substrate for CPO in accord with a proposed model for the active site of this enzyme. In addition, harderoporphyrinogen-VII, the monovinyl intermediate in the metabolism of Coproporphyrinogen-IV, was shown to be an equally good substrate for this enzyme. However, isoharderoporphyrinogen, which lacks the correct ordering of peripheral su...

  • role of aspartate 400 arginine 262 and arginine 401 in the catalytic mechanism of human Coproporphyrinogen oxidase
    Protein Science, 2007
    Co-Authors: Jason R Stephenson, Jon A Friesen, Justin B. Morgenthaler, Julie A. Stacey, Timothy D Lash, Marjorie A Jones
    Abstract:

    Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on Coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, Coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the Km value for Coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher Km for Coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was ∼3% compared to wild-type CPO, with a threefold increase in the Km value for Coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of Coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.

  • role of aspartate 400 arginine 262 and arginine 401 in the catalytic mechanism of human Coproporphyrinogen oxidase
    Protein Science, 2007
    Co-Authors: Jason R Stephenson, Jon A Friesen, Justin B. Morgenthaler, Julie A. Stacey, Timothy D Lash, Marjorie A Jones
    Abstract:

    Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on Coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, Coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for Coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for Coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for Coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of Coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.

  • investigation of the catalytic and structural roles of conserved histidines of human Coproporphyrinogen oxidase using site directed mutagenesis
    Medical Science Monitor, 2006
    Co-Authors: Shani J Gitter, Jon A Friesen, Christopher L. Cooper, Timothy D Lash, Marjorie A Jones
    Abstract:

    BACKGROUND: The catalytic contribution of four conserved histidines of human Coproporphyrinogen oxidase (CPO) has been investigated using site-directed mutagenesis to change histidine (H) into alanine (A). MATERIAL/METHODS: The wild-type and mutant enzyme forms were analyzed for their ability to utilize Coproporphyrinogen-III, mesoporphyrinogen-VI, and harderoporphyrinogen as substrates. RESULTS: Wild-type CPO had specific activities of 4.9+/-0.9 nmole product/min/mg for Coproporphyrinogen-III, 1.7+/-0.7 nmole product/min/mg for mesoporphyrinogen-VI, and 5.1+/-1.8 nmole product/min/mg for harderoporphyrinogen. The four mutant enzymes were catalytically competent with all three substrates, but to varying degrees. The most affected mutant was the H158A enzyme which exhibited approximately 50-fold lower activity than wild-type recombinant CPO. CONCLUSIONS: Thus, His158 of human CPO may have a role in the active site, but none of the conserved histidine residues of human Coproporphyrinogen oxidase is essential for catalytic activity although changes in histidines have been implicated in the disease state hereditary coproporphyria.

  • kinetic evaluation of human cloned Coproporphyrinogen oxidase using a ring isomer of the natural substrate
    Medical Science Monitor, 2005
    Co-Authors: Christopher L. Cooper, Timothy D Lash, Marjorie A Jones
    Abstract:

    BACKGROUND: The enzyme Coproporphyrinogen oxidase (copro'gen oxidase) converts Coproporphyrinogen-III (GIII) to protoporphyrinogen-IX via an intermediary monovinyl porphyrinogen. The A ring isomer Coproporphyrinogen-IV (C-IV) has previously been shown to be a substrate for copro'gen oxidase derived from avian erythrocytes. In contrast to the authentic substrate (C-III) where only a small amount of the monovinyl intermediate is detected, C-IV gives rise to a monovinyl intermediate that accumulates before being converted to an isomer of protoporphyrinogen-IX. No kinetic studies have been carried out using the purified human copro'gen oxidase to evaluate its ability to process both the authentic substrate as well as analogs. MATERIALS/METHODS: Therefore, purified, cloned human copro'gen oxidase was incubated with C-III or C-IV at 37 degrees C with various substrate concentrations (from 0.005 pM to 3.5 pM). The Km (an indication of molecular recognition) and Kcat (turnover number) values were determined. RESULTS: The Km value for total product formation was about the same with either C-III or C-IV indicating the same molecular recognition. However, the catalytic efficiency (Kcat/Km) of the enzyme for total product formation was not more than two fold higher using C-III relative to C-IV. CONCLUSIONS: Since the Km values are about the same for either substrate and the total Kcat/Km values are within two fold of each other, this could correlate with the increase of severity of porphyrias with monovinyl accumulation. The ability of the increased levels of C-IV to compete with the authentic substrate has important implications for clinical porphyrias.

Timothy D Lash - One of the best experts on this subject based on the ideXlab platform.

  • normal and abnormal heme biosynthesis 6 synthesis and metabolism of a series of monovinylporphyrinogens related to harderoporphyrinogen further insights into the oxidative decarboxylation of porphyrinogen substrates by Coproporphyrinogen oxidase
    Journal of Organic Chemistry, 2010
    Co-Authors: Timothy D Lash, Ukti N Mani, Annasigrid I M Keck, Marjorie A Jones
    Abstract:

    A series of vinylporphyrinogens were prepared to probe the enzyme Coproporphyrinogen oxidase (CPO). Six (2-chloroethyl)porphyrins were synthesized from a common dipyrrylmethane via a,c-biladiene intermediates in excellent yields. Subsequent dehydrohalogenation with DBU in refluxing DMF then gave the required vinylporphyrin methyl esters, including harderoporphyrin-I, harderoporphyrin-III, and isoharderoporphyrin. The corresponding porphyrinogen carboxylic acids were incubated with chicken red cell hemolysates, which contain the enzyme CPO, and the products analyzed. The 17-ethyl analogue of harderoporphyrinogen-III, but not its 13-ethyl isomer, was shown to be an excellent substrate for CPO in accord with a proposed model for the active site of this enzyme. In addition, harderoporphyrinogen-VII, the monovinyl intermediate in the metabolism of Coproporphyrinogen-IV, was shown to be an equally good substrate for this enzyme. However, isoharderoporphyrinogen, which lacks the correct ordering of peripheral su...

  • role of aspartate 400 arginine 262 and arginine 401 in the catalytic mechanism of human Coproporphyrinogen oxidase
    Protein Science, 2007
    Co-Authors: Jason R Stephenson, Jon A Friesen, Justin B. Morgenthaler, Julie A. Stacey, Timothy D Lash, Marjorie A Jones
    Abstract:

    Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on Coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, Coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the Km value for Coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher Km for Coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was ∼3% compared to wild-type CPO, with a threefold increase in the Km value for Coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of Coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.

  • role of aspartate 400 arginine 262 and arginine 401 in the catalytic mechanism of human Coproporphyrinogen oxidase
    Protein Science, 2007
    Co-Authors: Jason R Stephenson, Jon A Friesen, Justin B. Morgenthaler, Julie A. Stacey, Timothy D Lash, Marjorie A Jones
    Abstract:

    Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on Coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, Coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for Coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for Coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for Coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of Coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.

  • investigation of the catalytic and structural roles of conserved histidines of human Coproporphyrinogen oxidase using site directed mutagenesis
    Medical Science Monitor, 2006
    Co-Authors: Shani J Gitter, Jon A Friesen, Christopher L. Cooper, Timothy D Lash, Marjorie A Jones
    Abstract:

    BACKGROUND: The catalytic contribution of four conserved histidines of human Coproporphyrinogen oxidase (CPO) has been investigated using site-directed mutagenesis to change histidine (H) into alanine (A). MATERIAL/METHODS: The wild-type and mutant enzyme forms were analyzed for their ability to utilize Coproporphyrinogen-III, mesoporphyrinogen-VI, and harderoporphyrinogen as substrates. RESULTS: Wild-type CPO had specific activities of 4.9+/-0.9 nmole product/min/mg for Coproporphyrinogen-III, 1.7+/-0.7 nmole product/min/mg for mesoporphyrinogen-VI, and 5.1+/-1.8 nmole product/min/mg for harderoporphyrinogen. The four mutant enzymes were catalytically competent with all three substrates, but to varying degrees. The most affected mutant was the H158A enzyme which exhibited approximately 50-fold lower activity than wild-type recombinant CPO. CONCLUSIONS: Thus, His158 of human CPO may have a role in the active site, but none of the conserved histidine residues of human Coproporphyrinogen oxidase is essential for catalytic activity although changes in histidines have been implicated in the disease state hereditary coproporphyria.

  • kinetic evaluation of human cloned Coproporphyrinogen oxidase using a ring isomer of the natural substrate
    Medical Science Monitor, 2005
    Co-Authors: Christopher L. Cooper, Timothy D Lash, Marjorie A Jones
    Abstract:

    BACKGROUND: The enzyme Coproporphyrinogen oxidase (copro'gen oxidase) converts Coproporphyrinogen-III (GIII) to protoporphyrinogen-IX via an intermediary monovinyl porphyrinogen. The A ring isomer Coproporphyrinogen-IV (C-IV) has previously been shown to be a substrate for copro'gen oxidase derived from avian erythrocytes. In contrast to the authentic substrate (C-III) where only a small amount of the monovinyl intermediate is detected, C-IV gives rise to a monovinyl intermediate that accumulates before being converted to an isomer of protoporphyrinogen-IX. No kinetic studies have been carried out using the purified human copro'gen oxidase to evaluate its ability to process both the authentic substrate as well as analogs. MATERIALS/METHODS: Therefore, purified, cloned human copro'gen oxidase was incubated with C-III or C-IV at 37 degrees C with various substrate concentrations (from 0.005 pM to 3.5 pM). The Km (an indication of molecular recognition) and Kcat (turnover number) values were determined. RESULTS: The Km value for total product formation was about the same with either C-III or C-IV indicating the same molecular recognition. However, the catalytic efficiency (Kcat/Km) of the enzyme for total product formation was not more than two fold higher using C-III relative to C-IV. CONCLUSIONS: Since the Km values are about the same for either substrate and the total Kcat/Km values are within two fold of each other, this could correlate with the increase of severity of porphyrias with monovinyl accumulation. The ability of the increased levels of C-IV to compete with the authentic substrate has important implications for clinical porphyrias.

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

  • 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, Katrin Grage, Daniela Breckau, Peter Heathcote, Martina Jahn, Thomas Teschner, Volker Schunemann, Ava Masoumi, 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.

  • 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.

  • oxygen independent Coproporphyrinogen iii oxidase hemn from escherichia coli
    Journal of Biological Chemistry, 2002
    Co-Authors: Gunhild Layer, Esther Mahlitz, Knut Verfurth, Dieter Jahn
    Abstract:

    Abstract In bacteria the oxygen-independent Coproporphyrinogen-III oxidase catalyzes the oxygen-independent conversion of Coproporphyrinogen-III to protoporphyrinogen-IX. TheEscherichia coli hemN gene encoding a putative part of this enzyme was overexpressed in E. coli. Anaerobically purified HemN is a monomeric protein with a nativeM r = 52,000 ± 5,000. A newly established anaerobic enzyme assay was used to demonstrate for the first timein vitro Coproporphyrinogen-III oxidase activity for recombinant purified HemN. The enzyme requiresS-adenosyl-l-methionine (SAM), NAD(P)H, and additional cytoplasmatic components for catalysis. An oxygen-sensitive iron-sulfur cluster was identified by absorption spectroscopy and iron analysis. Cysteine residues Cys62, Cys66, and Cys69, which are part of the conserved CXXXCXXC motif found in all HemN proteins, are essential for iron-sulfur cluster formation and enzyme function. Completely conserved residues Tyr56 and His58, localized closely to the cysteine-rich motif, were found to be important for iron-sulfur cluster integrity. Mutation of Gly111 and Gly113, which are part of the potential GGGTP S-adenosyl-l-methionine binding motif, completely abolished enzymatic function. Observed functional properties in combination with a recently published computer-based enzyme classification (Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and Miller, N. E. (2001)Nucleic Acids Res. 29, 1097–1106) identifies HemN as “Radical SAM enzyme.” An appropriate enzymatic mechanism is suggested.

  • cloning and characterization of the escherichia coli hemn gene encoding the oxygen independent Coproporphyrinogen iii oxidase
    Journal of Bacteriology, 1995
    Co-Authors: B Troup, C Hungerer, Dieter Jahn
    Abstract:

    Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of Coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different Coproporphyrinogen III oxidases, one for aerobic (HemF) and one for anaerobic (HemN) conditions. Here we report the cloning of the hemN gene encoding the oxygen-independent Coproporphyrinogen III oxidase from Escherichia coli by complementation of a Salmonella typhimurium hemF hemN double mutant. An open reading frame of 1,371 bp encoding a protein of 457 amino acids with a calculated molecular mass of 52.8 kDa was identified. Sequence comparisons revealed 92% amino acid sequence identity to the recently cloned S. typhimurium hemN gene and 35% identity to the Rhodobacter sphaeroides gene. The hemN gene was mapped to 87.3 min of the E. coli chromosome and found identical to open reading frame o459 previously discovered during the genome sequencing project. Complementation of S. typhimurium hemF hemN double mutants with the E. coli hemN gene was detected under aerobic and anaerobic conditions, indicating an aerobic function for HemN. The previously cloned E. coli hemF gene encoding the oxygen-dependent enzyme complemented exclusively under aerobic conditions. Primer extension experiments revealed a strong transcription initiation site 102 bp upstream of the translational start site. DNA sequences with homology to a sigma 70-dependent promoter were detected. Expression of the hemN gene in response to changing environmental conditions was evaluated by using lacZ reporter gene fusions. Under anaerobic conditions, hemN expression was threefold greater than under aerobic growth conditions. Removal of iron from the growth medium resulted in an approximately fourfold decrease of aerobic hemN expression. Subsequent addition of iron restored normal expression.

  • isolation of the hemf operon containing the gene for the escherichia coli aerobic Coproporphyrinogen iii oxidase by in vivo complementation of a yeast hem13 mutant
    Journal of Bacteriology, 1994
    Co-Authors: B Troup, C Hungerer, Martina Jahn, Dieter Jahn
    Abstract:

    Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of Coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different Coproporphyrinogen III oxidases, one for aerobic and one for anaerobic conditions. Here we report the cloning of the hemF gene, encoding the aerobic Coproporphyrinogen III oxidase from Escherichia coli, by functional complementation of a Saccharomyces cerevisiae HEM13 mutant. An open reading frame of 897 bp encoding a protein of 299 amino acids with a calculated molecular mass of 34.3 kDa was identified. Sequence comparisons revealed 43% amino acid sequence identity with the product of the S. cerevisiae HEM13 gene and 90% identity with the product of the recently cloned Salmonella typhimurium hemF gene, while a structural relationship to the proposed anaerobic enzyme from Rhodobacter sphaeroides was not obvious. The hemF gene is in an operon with an upstream open reading frame (orf1) encoding a 31.7-kDa protein with homology to an amidase involved in cell wall metabolism. The hemF gene was mapped to 52.6 min of the E. coli chromosome. Primer extension experiments revealed a strong transcription initiation site upstream of orf1. A weak signal, possibly indicative of a second promoter, was also identified just upstream of the hemF gene. A region containing bent DNA (Bent 111), previously mapped to 52.6 min of the E. coli chromosome, was discovered in the 59 region of orf1. Two potential integration host factor binding sites were found, one close to each transcription start site. An open reading frame (orf3) transcribed in a direction opposite that of the hemF gene was found downstream of the hemF gene. It encodes a protein of 40.2 kDa that showed significant homology to proteins of the XylS/AraC family of transcriptional regulators. Images

Bernard Grandchamp - One of the best experts on this subject based on the ideXlab platform.

  • Molecular abnormalities of Coproporphyrinogen oxidase in patients with hereditary coproporphyria
    Journal of Bioenergetics and Biomembranes, 1995
    Co-Authors: Bernard Grandchamp, Jerome Lamoril
    Abstract:

    Genetic defects of Coproporphyrinogen oxidase (CPO) lead to hereditary coproporphyria, an inherited autosomal dominant porphyria. The recent cloning of human cDNAs and of the gene encoding CPO permits deducing the primary structure of the CPO protein and elucidating the molecular basis of HC in some families.

  • a molecular defect in Coproporphyrinogen oxidase gene causing harderoporphyria a variant form of hereditary coproporphyria
    Human Molecular Genetics, 1995
    Co-Authors: Jerome Lamoril, Vasco Da Silva, Jean-charles Deybach, Bernard Grandchamp, Pavel Martásek, Yves Nordmann
    Abstract:

    Hereditary coproporphyria (HC) is an acute hepatic porphyria with autosomal dominant inheritance caused by a deficient activity of Coproporphyrinogen IX oxidase (CPX). We previously described harderoporphyria, a homozygous variant form of coproporphyria in three siblings, characterized by a massive excretion of harderoporphyrin and a marked decrease of Coproporphyrinogen IX oxidase activity. In this kindred, the transmission of the disease was autosomal recessive. In the present study, sequencing of cDNA and genomic DNA from these patients revealed a point mutation resulting in a lysine to glutamic acid substitution (K304E) in exon 6 of the gene and the absence of the normal allele, suggesting a homozygous state for the mutation. Expression studies of normal and mutated cDNAs in E.coli demonstrated that this amino acid substitution was responsible for the important decrease in the enzyme activity and for the accumulation of harderoporphyrin. The Michaelis constant of the mutated enzyme was 10-fold higher than normal suggesting that the lysine at position 304 is important for binding the substrate: a slightly increased sensitivity to thermal denaturation was also observed

  • localization of the human Coproporphyrinogen oxidase gene to chromosome band 3q12
    Human Genetics, 1994
    Co-Authors: Valère Cacheux, Marie Helene Delfau, Luc Druart, Francoise Fougerousse, Gérard Tachdjian, Pavel Martásek, Bernard Grandchamp
    Abstract:

    The human gene encoding Coproporphyrinogen oxidase is the defective gene in hereditary coproporphyria. This gene was mapped to chromosome band 3q12 using fluorescent in situ hybridization. The chromosomal localization was confirmed by cosegregation of the human gene with chromosome 3 in a panel of human/rodent somatic hybrids.

  • molecular cloning sequencing and functional expression of a cdna encoding human Coproporphyrinogen oxidase
    Proceedings of the National Academy of Sciences of the United States of America, 1994
    Co-Authors: Pavel Martásek, J B Dumas, J J Montagne, Jean-michel Camadro, Hubert De Verneuil, Marie Helene Delfaularue, Pierre Labbe, Bernard Grandchamp
    Abstract:

    Abstract Coproporphyrinogen oxidase (EC 1.3.3.3) catalyzes the sixth step in the heme biosynthetic pathway, the oxidation of Coproporphyrinogen III to protoporphyrinogen IX. The activity of this enzyme is deficient in the disease hereditary coproporphyria. The sequence of the cDNA and predicted amino acid sequence of the human Coproporphyrinogen oxidase are presented. The human protein sequence contains a region completely homologous to that we obtained by sequencing an 11-amino acid peptide fragment from purified murine liver Coproporphyrinogen oxidase. Results of Southern blotting were consistent with the presence of a single human Coproporphyrinogen oxidase gene, and Northern blotting demonstrated one transcript of similar size in erythroid and nonerythroid cell lines. Expression of the cDNA coding for the putative mature human Coproporphyrinogen oxidase in Escherichia coli resulted in a 17-fold increase in Coproporphyrinogen activity over endogenous activity.

  • homozygous hereditary coproporphyria caused by an arginine to tryptophane substitution in Coproporphyrinogen oxidase and common intragenic polymorphisms
    Human Molecular Genetics, 1994
    Co-Authors: Pavel Martásek, Yves Nordmann, Bernard Grandchamp
    Abstract:

    Coproporphyrinogen oxidase is a mitochondrial hemebiosynthetic enzyme that converts Coproporphyrinogen to protoporphyrinogen. Inherited deficiency of this enzyme causes the human genetic disease hereditary coproporphyria. Recently, we isolated, sequenced and expressed the cDNA encoding human Coproporphyrinogen oxidase. This allowed us to investigate the nature of the defect leading to a profound deficiency of Coproporphyrinogen oxidase in a patient with homozygous hereditary coproporphyria. Using reversetranscription, amplification of the cDNA and direct sequencing of the amplified products, we found a point mutation resulted in an arginine to tryptophane substitution (R231W). Expression studies of normal and mutated cDNAs in a bacterial system demonstrated that this substitution resulted in the synthesis of an unstable protein with a residual catalytic activity

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, Claudia Noll, Hans Martin Schiebel, Dorit Kemken, Thomas Dulcks, Markus Kalesse, Dirk W Heinz, 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, Katrin Grage, Daniela Breckau, Peter Heathcote, Martina Jahn, Thomas Teschner, Volker Schunemann, Ava Masoumi, 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.

  • 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.

  • oxygen independent Coproporphyrinogen iii oxidase hemn from escherichia coli
    Journal of Biological Chemistry, 2002
    Co-Authors: Gunhild Layer, Esther Mahlitz, Knut Verfurth, Dieter Jahn
    Abstract:

    Abstract In bacteria the oxygen-independent Coproporphyrinogen-III oxidase catalyzes the oxygen-independent conversion of Coproporphyrinogen-III to protoporphyrinogen-IX. TheEscherichia coli hemN gene encoding a putative part of this enzyme was overexpressed in E. coli. Anaerobically purified HemN is a monomeric protein with a nativeM r = 52,000 ± 5,000. A newly established anaerobic enzyme assay was used to demonstrate for the first timein vitro Coproporphyrinogen-III oxidase activity for recombinant purified HemN. The enzyme requiresS-adenosyl-l-methionine (SAM), NAD(P)H, and additional cytoplasmatic components for catalysis. An oxygen-sensitive iron-sulfur cluster was identified by absorption spectroscopy and iron analysis. Cysteine residues Cys62, Cys66, and Cys69, which are part of the conserved CXXXCXXC motif found in all HemN proteins, are essential for iron-sulfur cluster formation and enzyme function. Completely conserved residues Tyr56 and His58, localized closely to the cysteine-rich motif, were found to be important for iron-sulfur cluster integrity. Mutation of Gly111 and Gly113, which are part of the potential GGGTP S-adenosyl-l-methionine binding motif, completely abolished enzymatic function. Observed functional properties in combination with a recently published computer-based enzyme classification (Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and Miller, N. E. (2001)Nucleic Acids Res. 29, 1097–1106) identifies HemN as “Radical SAM enzyme.” An appropriate enzymatic mechanism is suggested.