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Marjorie A Jones - One of the best experts on this subject based on the ideXlab platform.
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normal and abnormal heme biosynthesis part 7 synthesis and metabolism of Coproporphyrinogen iii analogues with acetate or butyrate side chains on rings c and d development of a modified model for the active site of Coproporphyrinogen Oxidase
Bioorganic & Medicinal Chemistry, 2011Co-Authors: Timothy D Lash, Teresa R Lamm, Andy J Schaber, Wenhsiang Chung, Eric K Johnson, Marjorie A JonesAbstract:Abstract Analogues of Coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H 2 SO 4 –TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9 . Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from Coproporphyrinogen Oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate Coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.
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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, 2010Co-Authors: Timothy D Lash, Annasigrid I M Keck, Ukti N Mani, Marjorie A JonesAbstract: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...
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Protective Effects of Covalent Cross-Linking on Proteolysis of Human Coproporphyrinogen Oxidase and Implications for Porphyria
American Journal of Biochemistry and Biotechnology, 2008Co-Authors: Ahmed W. Jafri, Jason R Stephenson, Jon A Friesen, Justin B. Morgenthaler, Marjorie A JonesAbstract:The effects of covalent cross-linkers on the enzyme, Coproporphyrinogen Oxidase, had been previously studied but their role in protecting the enzyme from protease cleavage has not been evaluated. Therefore, we examined how the cross-linker bis (sulfosuccinimidyl) suberate (BS3) affects the ability of trypsin to digest purified, wild type recombinant human Coproporphyrinogen Oxidase and selected mutants. Following incubation, the apparent molecular weights of peptides were evaluated by SDS-PAGE and enzymatic activity was assessed by spectroscopy following HPLC. For both wild type and mutants, the results indicated that the cross-linker was indeed able to protect against trypsin digestion relative to the enzyme incubated with trypsin in the absence of the cross-linker. These data have implications for the episodic nature of porphyria.
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use of di and tripropionate substrate analogs to probe the active site of human recombinant Coproporphyrinogen Oxidase
Medical Science Monitor, 2008Co-Authors: Justin B. Morgenthaler, Timothy D Lash, Reyna L Barto, Marjorie A JonesAbstract:Background: Defects in the enzyme Coproporphyrinogen Oxidase result in accumulation of porphyrins which may affect the severity of a subset of porphyrias. Thus evaluation of this enzyme for substrate selectivity is of value. Kinetic evaluations of recombinant human Coproporphyrinogen Oxidase have been undertaken using six di- and tripropionate analogs of the natural substrate coproporphyrin-ogen-III. These substrate analogs were modified by having alkyl groups in place of one or both of the ring 13- or 17-propionate moieties. Material/Methods: Cloned human enzyme was incubated with analogs under apparent first order conditions and with various substrate concentrations. The kinetic values, K m and V max , were determined. Results: Relative to the authentic substrate, the K m values for the 13-ethyl, dimethyl and diethyl porphyrinogens were very comparable whereas the K m values were much higher using dipropyl and dibutyl porphyrinogen and much lower for the 17-ethyl analog. For the dipropionate analogs, the V max values were an apparent function of the carbon length of the substituent on the C and D rings, with longer carbon length severely reducing product formation by some 4-5 orders of magnitude. Also, the two isomeric tripropionates that were tested indicated that it was more detrimental to have an ethyl group at the 13-position for both binding and catalysis. Conclusions: This work extends our understanding of porphyrin ring substituent effects reported by Cooper et al. (2005). The substituents on both the C and D rings have significant effects on both the substrate binding and catalysis by this important enzyme.
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role of aspartate 400 arginine 262 and arginine 401 in the catalytic mechanism of human Coproporphyrinogen Oxidase
Protein Science, 2007Co-Authors: Jason R Stephenson, Jon A Friesen, Timothy D Lash, Justin B. Morgenthaler, Julie A. Stacey, Marjorie A JonesAbstract: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.
Timothy D Lash - One of the best experts on this subject based on the ideXlab platform.
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normal and abnormal heme biosynthesis part 7 synthesis and metabolism of Coproporphyrinogen iii analogues with acetate or butyrate side chains on rings c and d development of a modified model for the active site of Coproporphyrinogen Oxidase
Bioorganic & Medicinal Chemistry, 2011Co-Authors: Timothy D Lash, Teresa R Lamm, Andy J Schaber, Wenhsiang Chung, Eric K Johnson, Marjorie A JonesAbstract:Abstract Analogues of Coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H 2 SO 4 –TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9 . Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from Coproporphyrinogen Oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate Coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.
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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, 2010Co-Authors: Timothy D Lash, Annasigrid I M Keck, Ukti N Mani, Marjorie A JonesAbstract: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...
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use of di and tripropionate substrate analogs to probe the active site of human recombinant Coproporphyrinogen Oxidase
Medical Science Monitor, 2008Co-Authors: Justin B. Morgenthaler, Timothy D Lash, Reyna L Barto, Marjorie A JonesAbstract:Background: Defects in the enzyme Coproporphyrinogen Oxidase result in accumulation of porphyrins which may affect the severity of a subset of porphyrias. Thus evaluation of this enzyme for substrate selectivity is of value. Kinetic evaluations of recombinant human Coproporphyrinogen Oxidase have been undertaken using six di- and tripropionate analogs of the natural substrate coproporphyrin-ogen-III. These substrate analogs were modified by having alkyl groups in place of one or both of the ring 13- or 17-propionate moieties. Material/Methods: Cloned human enzyme was incubated with analogs under apparent first order conditions and with various substrate concentrations. The kinetic values, K m and V max , were determined. Results: Relative to the authentic substrate, the K m values for the 13-ethyl, dimethyl and diethyl porphyrinogens were very comparable whereas the K m values were much higher using dipropyl and dibutyl porphyrinogen and much lower for the 17-ethyl analog. For the dipropionate analogs, the V max values were an apparent function of the carbon length of the substituent on the C and D rings, with longer carbon length severely reducing product formation by some 4-5 orders of magnitude. Also, the two isomeric tripropionates that were tested indicated that it was more detrimental to have an ethyl group at the 13-position for both binding and catalysis. Conclusions: This work extends our understanding of porphyrin ring substituent effects reported by Cooper et al. (2005). The substituents on both the C and D rings have significant effects on both the substrate binding and catalysis by this important enzyme.
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role of aspartate 400 arginine 262 and arginine 401 in the catalytic mechanism of human Coproporphyrinogen Oxidase
Protein Science, 2007Co-Authors: Jason R Stephenson, Jon A Friesen, Timothy D Lash, Justin B. Morgenthaler, Julie A. Stacey, Marjorie A JonesAbstract: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.
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Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human Coproporphyrinogen Oxidase
Protein Science, 2007Co-Authors: Jason R Stephenson, Jon A Friesen, Timothy D Lash, Justin B. Morgenthaler, Julie A. Stacey, Marjorie A JonesAbstract: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
Shigeru Taketani - One of the best experts on this subject based on the ideXlab platform.
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dual gene defects involving δ aminolaevulinate dehydratase and Coproporphyrinogen Oxidase in a porphyria patient
British Journal of Haematology, 2006Co-Authors: Shigeru Taketani, John D Phillips, Karl E Anderson, Reiko Akagi, Rikako Inoue, Shikibu Muranaka, Tsuyoshi Tahara, Shigeru SassaAbstract:Summary A Caucasian male had symptoms of acute porphyria, with increases in urinary δ-aminolaevulinic acid (ALA), porphobilinogen (PBG) and coproporphyrin that were consistent with hereditary coproporphyria (HCP). However, a greater than expected increase in ALA, compared with PBG, and a substantial increase in erythrocyte zinc protoporphyrin, suggested additional ALA dehydratase (ALAD) deficiency. Nucleotide sequence analysis of Coproporphyrinogen Oxidase (CPO) cDNA of the patient, but not of the parents, revealed a novel nucleotide transition G835C, resulting in an amino acid change, G279R. The mutant CPO protein expressed in Escherichia coli was unstable, and produced about 5% of activity compared with the wild-type CPO. Erythrocyte ALAD activity was 32% of normal in the proband. Nucleotide sequence analysis of cloned ALAD cDNAs from the patient revealed a C36G base transition (F12L amino acid change). The F12L ALAD mutation, which was found in the mother and a brother, was previously described, and is known to lack any enzyme activity. This patient thus represents the first case of porphyria where both CPO and ALAD deficiencies were demonstrated at the molecular level.
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expression of Coproporphyrinogen Oxidase and synthesis of hemoglobin in human erythroleukemia k562 cells
FEBS Journal, 2001Co-Authors: Shigeru Taketani, Takako Furukawa, Kazumichi FuruyamaAbstract:Coproporphyrinogen Oxidase (CPOX), the sixth enzyme in the heme-biosynthetic pathway, catalyzes oxidative decarboxylation of Coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA-transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with δ-aminolevulinic acid (ALA), most became benzidine-positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA-treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme-biosynthetic enzymes, including CPOX, ferrochelatase and erythroid-specific δ-aminolevulinic acid synthase, as well as of α-globin synthesis, were observed when cells were treated with transforming growth factor (TGF)β1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGFβ1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGFβ1-treated K562 cells led to a decrease in heme production compared with sense oligonucleotide-transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.
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cloning of a Coproporphyrinogen Oxidase promoter regulatory element binding protein
Biochemical and Biophysical Research Communications, 2000Co-Authors: Shinichiro Takahashi, Shigeru Taketani, Kazumichi Furuyama, Akira Kobayashi, Hideo Harigae, Masayuki Yamamoto, Kazuhiko Igarashi, Takeshi Sasaki, Norio HayashiAbstract:Coproporphyrinogen Oxidase [CPO] gene promoter regulatory element (CPRE) plays an important role in CPO gene regulation. To isolate a CPRE binding protein, we performed Southwestern screening of K562 cDNA expression library using CPRE as a probe and isolated a cDNA clone which encoded a novel protein, Klp1 (K562 cell-derived leucine-zipper-like protein 1). Klp1 mRNA was highly expressed in K562 cells, HeLa cells, and brain as a single transcript (1.4 kb). Gel mobility shift assays revealed that Klp1 specifically binds to CPRE. Computational analysis revealed that Klp1 has a leucine-zipper-like structure, a Leu-X-X-Leu-Leu motif, and a putative nuclear localization signal in the basic amino acid rich region. Transfection of the Klp1 expression vector into THP-1 cells resulted in transcriptional activation of a reporter construct containing CPRE. These results indicate that Klp1 is a DNA sequence-specific transcription factor that regulates gene expression of genes that contain CPRE in their regulatory region.
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oxidation of protoporphyrinogen ix in escherichia coli is mediated by the aerobic Coproporphyrinogen Oxidase
Molecular Genetics and Genomics, 1999Co-Authors: Shinichiro Narita, Shigeru Taketani, Hachiro InokuchiAbstract:Protoporphyrinogen Oxidase, the penultimate enzyme involved in the biosynthetic pathway for heme, catalyzes the removal of six electrons from protoporphyrinogen IX to generate protoporphyrin IX. In Escherichia coli, this enzyme is encoded by the hemG gene. In this study we examined possible alternate pathways for the oxidation of protoporphyrinogen IX to protoporphyrin IX, by isolating and investigating E. coli mutants that can still grow normally when the hemG gene is disrupted. One of these mutants was characterized in detail and had a mutation in the promoter region of the hemF gene, which encodes aerobic Coproporphyrinogen Oxidase, the enzyme involved in the step immediately before protoporphyrinogen Oxidase. Measurement of the promoter activity of the hemF gene showed that the level of transcription was elevated by the mutation. Overexpression of a wild-type hemF gene cloned in a multicopy plasmid also restored the growth of ΔhemG strain. Extracts from cells that overexpress hemF exhibited an increased ability to oxidize protoporphyrinogen IX to protoporphyrin IX. These findings suggest that the E. coli aerobic Coproporphyrinogen Oxidase has an intrinsic capacity to oxidize not only Coproporphyrinogen III but also protoporphyrinogen IX.
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differential regulation of Coproporphyrinogen Oxidase gene between erythroid and nonerythroid cells
Blood, 1998Co-Authors: Shinichiro Takahashi, Shigeru Taketani, Akira Kobayashi, Masayuki Yamamoto, Norio Hayashi, Junetsu Akasaka, Tadashi NagaiAbstract:Coproporphyrinogen Oxidase (CPO) catalyzes the sixth step of the heme biosynthetic pathway. To assess the tissue-specific regulation of the CPO gene promoter, mouse genomic DNA clones for CPO were isolated. Structural analysis demonstrated that the mouse CPO gene spans approximately 11 kb and consists of seven exons, just like its human counterpart. Functional analysis of the promoter by transient transfection assays indicated that synergistic action between an SP-1–like element at −21/−12, a GATA site at −59/−54, and a novel regulatory element, CPRE (-GGACTACAG-) at −49/−41, is essential for the promoter activity in murine erythroleukemia (MEL) cells. In nonerythroid NIH3T3 cells, however, the GATA site is not required. Gel mobility shift assays demonstrated that specific DNA-protein complexes can be formed with each element, and that there are cell-specific differences in factors, which bind to the SP-1–like element between MEL and NIH3T3 cells. These results provide evidence for differential regulation of the promoter function of CPO gene between erythroid and nonerythroid cells. © 1998 by The American Society of Hematology.
Bernard Grandchamp - One of the best experts on this subject based on the ideXlab platform.
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Human Coproporphyrinogen Oxidase. Biochemical characterization of recombinant normal and R231W mutated enzymes expressed in E. coli as soluble, catalytically active homodimers.
Cellular and Molecular Biology, 1997Co-Authors: Pavel Martasek, Jean-michel Camadro, Bernard Grandchamp, Chander Raman, M. C. Lecomte, J. P. Le Caer, Borries Demeler, P LabbeAbstract:To obtain recombinant human Coproporphyrinogen Oxidase (CPX), a cDNA for the coding region of mature human CPX has been expressed in E. coli. CPX was produced as a fusion protein with glutathione S-transferase followed by the hexapeptide recognition site for thrombin cleavage just preceding first amino acid of the CPX protein. The human CPX was found to be in the soluble fraction. This previously unobtainable human heme synthetic enzyme was purified to electrophoretic homogeneity with a specific activity of 4200 nmol/hr./mg of protein using a Glutathione Sepharose 4B column and gel filtration. Recombinant human CPX exhibits homogeneous behavior during high performance liquid chromatography (HPLC) and the N-terminal sequence, confirmed by protein sequencing, revealed a single polypeptide chain. In its active form, human CPX is a homodimer. According to the hydrodynamic properties derived from analytical ultracentrifugation, dimeric CPX has a nearly globular shape. Additionally, naturally occurring Arg to Trp (R231W)-mutated CPX has been also expressed in E. coli and further characterized. The mutated enzyme has a Km value of 0.55 microM as compared to 0.30 microM for the wild type. The catalytic efficiency (specificity constant, kcat/Km) of the mutated CPX was four fold lower than wild-type enzyme. The activity measurement of the mutated enzyme showed higher thermal sensitivity as compared with wild type CPX. The measured pI for mutated CPX is 5.65, compared to 6.40 for wild type. The pH optima for the mutated and wild-type protein are 6.6 and 6.8, respectively. The R231W mutation of CPX does not affect dimer formation and both normal and mutated CPX exhibit identical sedimentation properties. The thermal denaturation of both wild type and mutant CPX was found to be irreversible. The mutated CPX contained a significant amount of tightly bound porphyrin coproporphyrin. No metal association was found either in wild type or in mutated CPX. The availability of the recombinant human CPX will aid in structural and mechanistic studies.
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Molecular abnormalities of Coproporphyrinogen Oxidase in patients with hereditary coproporphyria
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Bernard Grandchamp, Jerome LamorilAbstract: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.
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localization of the human Coproporphyrinogen Oxidase gene to chromosome band 3q12
Human Genetics, 1994Co-Authors: Valere Cacheux, Pavel Martasek, Francoise Fougerousse, Marie Helene Delfau, Luc Druart, Gerard Tachdjian, Bernard GrandchampAbstract: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.
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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, 1994Co-Authors: Pavel Martasek, J M Camadro, Mariehelene Delfaularue, J B Dumas, J J Montagne, H De Verneuil, P Labbe, Bernard GrandchampAbstract: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.
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homozygous hereditary coproporphyria caused by an arginine to tryptophane substitution in Coproporphyrinogen Oxidase and common intragenic polymorphisms
Human Molecular Genetics, 1994Co-Authors: Pavel Martasek, Y Nordmann, Bernard GrandchampAbstract: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
Kjeld A. Marcker - One of the best experts on this subject based on the ideXlab platform.
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A soybean Coproporphyrinogen Oxidase gene is highly expressed in root nodules
Plant Molecular Biology, 1993Co-Authors: Ole Madsen, Lene Sandal, Niels N. Sandal, Kjeld A. MarckerAbstract:In plants the enzyme Coproporphyrinogen Oxidase catalyzes the oxidative decarboxylation of Coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s). We have isolated a soybean Coproporphyrinogen Oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The Coproporphyrinogen Oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide. A full-length Coproporphyrinogen Oxidase cDNA clone complements a yeast mutant deleted of the Coproporphyrinogen Oxidase gene, thus demonstrating the function of the soybean protein. The soybean Coproporphyrinogen Oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The Coproporphyrinogen Oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots. The high level of Coproporphyrinogen Oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation.
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a soybean Coproporphyrinogen Oxidase gene is highly expressed in root nodules
Plant Molecular Biology, 1993Co-Authors: Ole Lehrmann Madsen, Lene Sandal, Niels N. Sandal, Kjeld A. MarckerAbstract:In plants the enzyme Coproporphyrinogen Oxidase catalyzes the oxidative decarboxylation of Coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s).