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James P Kushner - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for tetrapyrrole coordination by Uroporphyrinogen Decarboxylase
The EMBO Journal, 2003Co-Authors: John D Phillips, James P Kushner, Frank G. Whitby, Christopher P. HillAbstract:Uroporphyrinogen Decarboxylase (URO-D), an essential enzyme that functions in the heme biosynthetic pathway, catalyzes decarboxylation of all four acetate groups of Uroporphyrinogen to form coproporphyrinogen. Here we report crystal structures of URO-D in complex with the I and III isomer coproporphyrinogen products. Crystallization required use of a novel enzymatic approach to generate the highly oxygen-sensitive porphyrinogen substrate in situ. The tetrapyrrole product adopts a domed conformation that lies against a collar of conserved hydrophobic residues and allows formation of hydrogen bonding interactions between a carboxylate oxygen atom of the invariant Asp86 residue and the pyrrole NH groups. Structural and biochemical analyses of URO-D proteins mutated at Asp86 support the conclusion that this residue makes important contributions to binding and likely promotes catalysis by stabilizing a positive charge on a reaction intermediate. The central coordination geometry of Asp86 allows the initial substrates and the various partially decarboxylated intermediates to be bound with equivalent activating interactions, and thereby explains how all four of the substrate acetate groups can be decarboxylated at the same catalytic center.
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uroporphyria in the Uroporphyrinogen Decarboxylase deficient mouse interplay with siderosis and polychlorinated biphenyl exposure
Hepatology, 2002Co-Authors: Michael R Franklin, John D Phillips, James P KushnerAbstract:Several methods have been used to develop rodent models with the hepatic manifestations of porphyria cutanea tarda (PCT). Acute iron administration or mutations of the hemochromatosis gene (Hfe) have been used to generate hepatic siderosis, a nearly uniform finding in PCT. Heterozygosity for a null mutation at the Uroporphyrinogen Decarboxylase (Uro-D+/-) locus has been developed to mimic familial PCT in humans. This study examines the interplay of these 2 genetic risk factors and their influence, alone and combined with polychlorinated-biphenyl exposure. Neither an Hfe-null mutation nor iron-dextran administration alone or in combination with polychlorinated biphenyl exposure was porphyrinogenic in a 3-week model using mice wild-type at the Uro-D locus. Homozygosity for an Hfe-null mutation significantly elevated hepatic iron but not to the extent seen with parenteral iron-dextran administration. Homozygosity for an Hfe-null mutation but not iron-dextran administration was porphyrinogenic in animals heterozygous for the Uro-D mutation. Polychlorinated biphenyls were also porphyrinogenic in these animals. Uroporphyria in Uro-D+/- animals was exacerbated by combinations of the homozygous Hfe-null mutation and polychlorinated biphenyls and iron-dextran and polychlorinated biphenyls. In all cases in which uroporphyria developed, a greater degree of experimental uroporphyria was seen in female animals. All elevated hepatic uroporphyrin concentrations were accompanied by depressed Uroporphyrinogen Decarboxylase activity and the presence of a factor in cytosol that inhibits recombinant human Uroporphyrinogen Decarboxylase. In conclusion, the expression of the uroporphyric phenotype, dependent on the susceptibility imparted by a genetic mutation, provides a uniquely facile model for dissecting the molecular pathogenesis of the disease.
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measurement of Uroporphyrinogen Decarboxylase activity
Current protocols in immunology, 1999Co-Authors: John D Phillips, James P KushnerAbstract:Uroporphyrinogen Decarboxylase (UROD) catalyzes decarboxylation of the four acetate side chains of urophyrinogen to form coproporphyrinogen. Activity of UROD can be measured using an enzymatically prepared substrate or a chemically prepared one. For the former, bacterial porphobilinogen deaminase is prepared and used to prepare the porphyrinogen substrate for the enzymatic assay. Erythrocyte lysates can be used to measure hemoglobin content as an indicator of UROD activity.
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Current Protocols in Toxicology - Measurement of Uroporphyrinogen Decarboxylase Activity
Current protocols in immunology, 1999Co-Authors: John D Phillips, James P KushnerAbstract:Uroporphyrinogen Decarboxylase (UROD) catalyzes decarboxylation of the four acetate side chains of urophyrinogen to form coproporphyrinogen. Activity of UROD can be measured using an enzymatically prepared substrate or a chemically prepared one. For the former, bacterial porphobilinogen deaminase is prepared and used to prepare the porphyrinogen substrate for the enzymatic assay. Erythrocyte lysates can be used to measure hemoglobin content as an indicator of UROD activity.
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Crystal structure of human Uroporphyrinogen Decarboxylase
The EMBO Journal, 1998Co-Authors: Frank G. Whitby, James P Kushner, John D Phillips, Christopher P. HillAbstract:Uroporphyrinogen Decarboxylase (URO‐D) catalyzes the fifth step in the heme biosynthetic pathway, converting Uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. This activity is essential in all organisms, and subnormal activity of URO‐D leads to the most common form of porphyria in humans, porphyria cutanea tarda (PCT). We have determined the crystal structure of recombinant human URO‐D at 1.60 A resolution. The 40.8 kDa protein is comprised of a single domain containing a (β/α) 8 ‐barrel with a deep active site cleft formed by loops at the C‐terminal ends of the barrel strands. Many conserved residues cluster at this cleft, including the invariant side chains of Arg37, Arg41 and His339, which probably function in substrate binding, and Asp86, Tyr164 and Ser219, which may function in either binding or catalysis. URO‐D is a dimer in solution ( K d = 0.1 μM), and this dimer also appears to be formed in the crystal. Assembly of the dimer juxtaposes the active site clefts of the monomers, suggesting a functionally important interaction between the catalytic centers.
John D Phillips - One of the best experts on this subject based on the ideXlab platform.
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hepatoerythropoietic porphyria due to a novel mutation in the Uroporphyrinogen Decarboxylase gene
British Journal of Dermatology, 2011Co-Authors: Jordi Tofigueras, John D Phillips, J M Gonzalezlopez, C Badenas, Irene Madrigal, E M Gonzalezromaris, C Ramos, J M Aguirre, C HerreroAbstract:Background Hepatoerythropoietic porphyria (HEP) is a rare form of porphyria that results from a deficiency of Uroporphyrinogen Decarboxylase (UROD). The disease is caused by homoallelism or heteroallelism for mutations in the UROD gene.
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Structural basis for tetrapyrrole coordination by Uroporphyrinogen Decarboxylase
The EMBO Journal, 2003Co-Authors: John D Phillips, James P Kushner, Frank G. Whitby, Christopher P. HillAbstract:Uroporphyrinogen Decarboxylase (URO-D), an essential enzyme that functions in the heme biosynthetic pathway, catalyzes decarboxylation of all four acetate groups of Uroporphyrinogen to form coproporphyrinogen. Here we report crystal structures of URO-D in complex with the I and III isomer coproporphyrinogen products. Crystallization required use of a novel enzymatic approach to generate the highly oxygen-sensitive porphyrinogen substrate in situ. The tetrapyrrole product adopts a domed conformation that lies against a collar of conserved hydrophobic residues and allows formation of hydrogen bonding interactions between a carboxylate oxygen atom of the invariant Asp86 residue and the pyrrole NH groups. Structural and biochemical analyses of URO-D proteins mutated at Asp86 support the conclusion that this residue makes important contributions to binding and likely promotes catalysis by stabilizing a positive charge on a reaction intermediate. The central coordination geometry of Asp86 allows the initial substrates and the various partially decarboxylated intermediates to be bound with equivalent activating interactions, and thereby explains how all four of the substrate acetate groups can be decarboxylated at the same catalytic center.
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uroporphyria in the Uroporphyrinogen Decarboxylase deficient mouse interplay with siderosis and polychlorinated biphenyl exposure
Hepatology, 2002Co-Authors: Michael R Franklin, John D Phillips, James P KushnerAbstract:Several methods have been used to develop rodent models with the hepatic manifestations of porphyria cutanea tarda (PCT). Acute iron administration or mutations of the hemochromatosis gene (Hfe) have been used to generate hepatic siderosis, a nearly uniform finding in PCT. Heterozygosity for a null mutation at the Uroporphyrinogen Decarboxylase (Uro-D+/-) locus has been developed to mimic familial PCT in humans. This study examines the interplay of these 2 genetic risk factors and their influence, alone and combined with polychlorinated-biphenyl exposure. Neither an Hfe-null mutation nor iron-dextran administration alone or in combination with polychlorinated biphenyl exposure was porphyrinogenic in a 3-week model using mice wild-type at the Uro-D locus. Homozygosity for an Hfe-null mutation significantly elevated hepatic iron but not to the extent seen with parenteral iron-dextran administration. Homozygosity for an Hfe-null mutation but not iron-dextran administration was porphyrinogenic in animals heterozygous for the Uro-D mutation. Polychlorinated biphenyls were also porphyrinogenic in these animals. Uroporphyria in Uro-D+/- animals was exacerbated by combinations of the homozygous Hfe-null mutation and polychlorinated biphenyls and iron-dextran and polychlorinated biphenyls. In all cases in which uroporphyria developed, a greater degree of experimental uroporphyria was seen in female animals. All elevated hepatic uroporphyrin concentrations were accompanied by depressed Uroporphyrinogen Decarboxylase activity and the presence of a factor in cytosol that inhibits recombinant human Uroporphyrinogen Decarboxylase. In conclusion, the expression of the uroporphyric phenotype, dependent on the susceptibility imparted by a genetic mutation, provides a uniquely facile model for dissecting the molecular pathogenesis of the disease.
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measurement of Uroporphyrinogen Decarboxylase activity
Current protocols in immunology, 1999Co-Authors: John D Phillips, James P KushnerAbstract:Uroporphyrinogen Decarboxylase (UROD) catalyzes decarboxylation of the four acetate side chains of urophyrinogen to form coproporphyrinogen. Activity of UROD can be measured using an enzymatically prepared substrate or a chemically prepared one. For the former, bacterial porphobilinogen deaminase is prepared and used to prepare the porphyrinogen substrate for the enzymatic assay. Erythrocyte lysates can be used to measure hemoglobin content as an indicator of UROD activity.
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Current Protocols in Toxicology - Measurement of Uroporphyrinogen Decarboxylase Activity
Current protocols in immunology, 1999Co-Authors: John D Phillips, James P KushnerAbstract:Uroporphyrinogen Decarboxylase (UROD) catalyzes decarboxylation of the four acetate side chains of urophyrinogen to form coproporphyrinogen. Activity of UROD can be measured using an enzymatically prepared substrate or a chemically prepared one. For the former, bacterial porphobilinogen deaminase is prepared and used to prepare the porphyrinogen substrate for the enzymatic assay. Erythrocyte lysates can be used to measure hemoglobin content as an indicator of UROD activity.
Andrew G. Roberts - One of the best experts on this subject based on the ideXlab platform.
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Purification and properties of Uroporphyrinogen Decarboxylase from human erythrocytes.
Methods in Enzymology, 2004Co-Authors: Andrew G. Roberts, George H. ElderAbstract:Publisher Summary Uroporphyrinogen Decarboxylase (UROD) is a cytosolic enzyme of the heme biosynthetic pathway. This chapter discusses the purification and properties of Uroporphyrinogen Decarboxylase from human erythrocytes. Erythrocytes are, therefore, a convenient source from which homogeneous preparations of UROD can be obtained in relatively good yield. The enzyme has also been purified to homogeneity from chicken erythrocytes, bovine liver, yeast, and Rhodobacter spheroides . All stages of the purification procedure are carried out at 4°. Dithiothreitol (DTT) is added to all buffers immediately before the use to a final concentration of 0.5 m M . Uroporphyrinogen Decarboxylase activity is monitored at each stage of the purification procedure by measuring the conversion of pentacarboxylate porphyrinogen III to coproporphyrinogen III or by a modification in which porphyrins are not converted to their methyl esters but separated directly by reversed-phase high-performance liquid chromatography. For some preparations, UROD concentration is measured by electroimmunoassay, using a rabbit polyclonal antiserum to purified human erythrocyte UROD.
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co inheritance of mutations in the Uroporphyrinogen Decarboxylase and hemochromatosis genes accelerates the onset of porphyria cutanea tarda
Journal of Investigative Dermatology, 2000Co-Authors: Jennifer J Brady, G.h. Elder, Andrew G. Roberts, Rhian R Morgan, Sharon D Whatley, Gareth Rowlands, Rosemarie Watson, Helen A Jackson, Mark Worwood, Cindy M DarbyAbstract:Porphyria cutanea tarda is a skin disease caused by photosensitization by porphyrins whose accumulation is caused by deficiency of hepatic uroporphyrin- ogen Decarboxylase activity. Mutations in the Uroporphyrinogen Decarboxylase gene are present in the low-penetrant, autosomal dominant familial form but not in the commoner sporadic form of porphyria cutanea tarda. We have investigated the relationship between age of onset of skin lesions and mutations (C282Y, H63D) in the hemochromatosis gene in familial (19 patients) and sporadic porphyria cutanea tarda (65 patients). Familial porphyria cutanea tarda was identified by mutational analysis of the Uroporphyrinogen Decarboxylase gene. Five previously described and eight novel mutations (A80S, R144P, L216Q, E218K, L282R, G303S, 402–403delGT, IVS2 + 2 delTAA) were identified. Homozygosity for the C282Y hemochromatosis mutation was associated with an earlier onset of skin lesions in both familial and sporadic porphyria cutanea tarda, the effect being more marked in familial porphyria cutanea tarda where anticipation was demonstrated in family studies. Analysis of the frequencies of hemochromatosis genotypes in each type of porphyria cutanea tarda indicated that C282Y homozygosity is an important susceptibility factor in both types but suggested that heterozygosity for this mutation has much less effect on the development of the disease.
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a mutation g281e of the human Uroporphyrinogen Decarboxylase gene causes both hepatoerythropoietic porphyria and overt familial porphyria cutanea tarda biochemical and genetic studies on spanish patients
Journal of Investigative Dermatology, 1995Co-Authors: Andrew G. Roberts, George H. Elder, C Herrero, Rafael Enriques De Salamanca, Mario Lecha, J M MascaroAbstract:Hepatoerythropoietic porphyria is a severe cutaneous porphyria caused by deficiency of Uroporphyrinogen Decarboxylase and is considered to be the homozygous form of familial (type II) porphyria cutanea tarda. To elucidate further the relation between these conditions, we studied five Spanish families with hepatoerythropoietic porphyria and nine unrelated Spanish patients with familial porphyria cutanea tarda. Immunoreactive and catalytic Uroporphyrinogen Decarboxylase was decreased by greater than 95% in the five patients with hepatoerythropoietic porphyria. Hepatic Uroporphyrinogen Decarboxylase activity was decreased to 22% of normal. Four patients were homozygous for a mutation (G281E) originally identified in a Tunisian family; the fifth patient was a compound heterozygote for this mutation. The calculated carrier frequency for G281E in Spain is one in 1800. None of the nine familial porphyria cutanea tarda patients carried the G281E mutation. However, one G281E heterozygote in a family with hepatoerythropoietic porphyria had overt porphyria cutanea tarda. These findings suggest that the G281E mutation is functionally less severe than erythrocyte measurements indicate, that its clinical penetrance is very low in heterozygotes, and that, for this particular mutation, hepatoerythropoietic porphyria is the homozygous form of familial porphyria cutanea tarda.
Niels Erik Petersen - One of the best experts on this subject based on the ideXlab platform.
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Expression and characterization of six clinically relevant Uroporphyrinogen Decarboxylase gene mutations.
Scandinavian Journal of Clinical & Laboratory Investigation, 2009Co-Authors: L Christiansen, J. Brøns-poulsen, Mogens Hørder, Axel Brock, Niels Erik PetersenAbstract:The functional consequence of six Uroporphyrinogen Decarboxylase (UROD) gene mutations found in Danish patients with familial porphyria cutanea tarda was investigated. Wild-type UROD and the 6 mutants (3 missense, 1 nonsense and 2 frameshift mutants) were cloned and expressed using the prokaryotic gGEX-6P system, in which the protein is produced in fusion with glutathione S-transferase (GST). Enzymatic activity of the purified recombinant mutant fusion proteins ranged from undetectable to less than 12% of the recombinant wild-type protein. Mutant proteins cleaved from the GST part did not retain any catalytic activity. These observations can be ascribed to the structure/function relationships of the enzyme, and the fact that the enzyme is a dimer in its active form. Although the clinical manifestation of familial porphyria cutanea tarda is complex, the findings support the notion that different mutations may affect individuals differently.
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Uroporphyrinogen Decarboxylase gene mutations in danish patients with porphyria cutanea tarda
Scandinavian Journal of Clinical & Laboratory Investigation, 2000Co-Authors: L Christiansen, Anette Bygum, A Jensen, F Brandrup, K Thomsen, M Horder, Niels Erik PetersenAbstract:Decreased Uroporphyrinogen Decarboxylase (UROD) activity is a characteristic feature of the most common of the porphyrias, porphyria cutanea tarda (PCT). A subgroup of the clinically overt PCT cases is associated with mutations in the gene encoding UROD and inherited as an autosomal-dominant trait. In this study, DNAs from 53 Danish PCT patients were subjected to genetic analysis for UROD mutations using denaturing gradient gel electrophoresis. Eleven genetic variations, seven of which are possible disease causing, were identified. All but one of these mutations were previously unknown, lending further support to the assumption that PCT is a heteroallelic disease. Only 11% of the examined patients were previously recognized as familial PCT cases. However, possible disease-related UROD mutations were identified in 24% of the examined patients, indicating that genetic analysis of PCT patients may improve differentiation between familial and sporadic PCT cases.
C Herrero - One of the best experts on this subject based on the ideXlab platform.
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hepatoerythropoietic porphyria due to a novel mutation in the Uroporphyrinogen Decarboxylase gene
British Journal of Dermatology, 2011Co-Authors: Jordi Tofigueras, John D Phillips, J M Gonzalezlopez, C Badenas, Irene Madrigal, E M Gonzalezromaris, C Ramos, J M Aguirre, C HerreroAbstract:Background Hepatoerythropoietic porphyria (HEP) is a rare form of porphyria that results from a deficiency of Uroporphyrinogen Decarboxylase (UROD). The disease is caused by homoallelism or heteroallelism for mutations in the UROD gene.
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childhood onset mild cutaneous porphyria with compound heterozygotic mutations in the Uroporphyrinogen Decarboxylase gene
Clinical and Experimental Dermatology, 2008Co-Authors: E Remenyik, C Badenas, C Herrero, M Lecha, Ferenc Koszo, V Vass, V Varga, G Emri, A Balogh, I HorkayAbstract:Summary Three children (two boys and one girl) from the same family presented with photosensitivity, hyperpigmentation, hypertrichosis, mild skin fragility, blistering and scarring in childhood. On examination, the cutaneous lesions were found to have improved since their previous examinations. Laboratory tests showed raised plasma and urine carboxyporphyrins and decreased Uroporphyrinogen Decarboxylase enzyme activity in red blood cells. Triggering factors for porphyria were not detected except for a hepatitis C virus infection in the younger boy. The girl's clinical symptoms recurred in late adolescence, after iron and oestrogen treatments. Mutation analysis of the UROD gene detected two missense mutations, 19 AG M1V (novel) and 703CT P235S (previously reported), in an uncommon compound heterozygous manner in the three siblings.
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a mutation g281e of the human Uroporphyrinogen Decarboxylase gene causes both hepatoerythropoietic porphyria and overt familial porphyria cutanea tarda biochemical and genetic studies on spanish patients
Journal of Investigative Dermatology, 1995Co-Authors: Andrew G. Roberts, George H. Elder, C Herrero, Rafael Enriques De Salamanca, Mario Lecha, J M MascaroAbstract:Hepatoerythropoietic porphyria is a severe cutaneous porphyria caused by deficiency of Uroporphyrinogen Decarboxylase and is considered to be the homozygous form of familial (type II) porphyria cutanea tarda. To elucidate further the relation between these conditions, we studied five Spanish families with hepatoerythropoietic porphyria and nine unrelated Spanish patients with familial porphyria cutanea tarda. Immunoreactive and catalytic Uroporphyrinogen Decarboxylase was decreased by greater than 95% in the five patients with hepatoerythropoietic porphyria. Hepatic Uroporphyrinogen Decarboxylase activity was decreased to 22% of normal. Four patients were homozygous for a mutation (G281E) originally identified in a Tunisian family; the fifth patient was a compound heterozygote for this mutation. The calculated carrier frequency for G281E in Spain is one in 1800. None of the nine familial porphyria cutanea tarda patients carried the G281E mutation. However, one G281E heterozygote in a family with hepatoerythropoietic porphyria had overt porphyria cutanea tarda. These findings suggest that the G281E mutation is functionally less severe than erythrocyte measurements indicate, that its clinical penetrance is very low in heterozygotes, and that, for this particular mutation, hepatoerythropoietic porphyria is the homozygous form of familial porphyria cutanea tarda.