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Oscar Millet - One of the best experts on this subject based on the ideXlab platform.
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natural and pharmacological chaperones against accelerated protein degradation Uroporphyrinogen III synthase and congenital erythropoietic porphyria
2020Co-Authors: Ganeko Bernardoseisdedos, Emmanuel Richard, David Gil, Jeanmarc Blouin, Oscar MilletAbstract:Abstract Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disease derived from a deficient activity in the fourth enzyme of the heme biosynthetic pathway, Uroporphyrinogen III synthase (UROIIIS). The impossibility of a correct heme production results in the accumulation of intermediate metabolites known as uroporhyrins, leading to pathological consequences such as skin photosensitivity and phototoxic cutaneous injuries. In this chapter, we discuss the importance of protein homeostasis in the heme pathway and its regulation, as well as how many of the CEP-causing mutations impair UROIIIS homeostasis, affecting thermodynamic stability but preserving normal or near-normal activity. As a proof of concept, we show how UROIIIS proteostasis can be restored by use of a proteasomal inhibitor in animal models. Finally, a new line of therapeutic intervention against CEP is presented, which involves the use of a pharmacological chaperone known as ciclopirox (CPX) to improve UROIIIS stability, reverting most of the CEP symptoms in mice models.
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tuning intracellular homeostasis of human Uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria
Human Molecular Genetics, 2014Co-Authors: Fredj Ben Bdira, Ana Lain, Esperanza Gonzalez, Juan M Falconperez, Paula Pluta, Arantza Sanzparra, Oscar MilletAbstract:Congenital erythropoietic porphyria (CEP) results from a deficiency in Uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.
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intracellular rescue of the Uroporphyrinogen III synthase activity in enzymes carrying the hotspot mutation c73r
Journal of Biological Chemistry, 2011Co-Authors: Arola Fortian, David Castano, Esperanza Gonzalez, Juan M Falconperez, Oscar MilletAbstract:A single mutation (C73R) in the enzyme Uroporphyrinogen III synthase (UROIIIS) is responsible for more than one-third of all of the reported cases of the rare autosomal disease congenital erythropoietic porphyria (CEP). CEP patients carrying this hotspot mutation develop a severe phenotype of the disease, including reduced life expectancy. Here, we have investigated the molecular basis for the functional deficit in the mutant enzyme both in vitro and in cellular systems. We show that a Cys in position 73 is not essential for the catalytic activity of the enzyme but its mutation to Arg speeds up the process of irreversible unfolding and aggregation. In the mammalian cell milieu, the mutant protein levels decrease to below the detection limit, whereas wild type UROIIIS can be detected easily. The disparate response is not produced by differences at the level of transcription, and the results with cultured cells and in vitro are consistent with a model where the protein becomes very unstable upon mutation and triggers a degradation mechanism via the proteasome. Mutant protein levels can be restored upon cell treatment with the proteasome inhibitor MG132. The intracellularly recovered C73R-UROIIIS protein shows enzymatic activity, paving the way for a new line of therapeutic intervention in CEP patients.
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structural thermodynamic and mechanistical studies in Uroporphyrinogen III synthase molecular basis of congenital erythropoietic porphyria
Advances in Protein Chemistry, 2011Co-Authors: Arola Fortian, Ana Lain, David Castano, Esperanza Gonzalez, Juan M Falconperez, Oscar MilletAbstract:Congenital erythropoietic porphyria (CEP) is a rare autosomal disease ultimately related to deleterious mutations in Uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the biosynthetic route of the heme group. UROIIIS catalyzes the cyclization of the linear tetrapyrrol hydroxymethylbilane (HMB), inverting the configuration in one of the aromatic rings. In the absence of the enzyme (or when ill-functioning), HMB spontaneously degrades to the by-product Uroporphyrinogen I, which cannot lead to the heme group and accumulates in the body, producing some of the symptoms observed in CEP patients. In the present chapter, clinical, biochemical, and biophysical information has been compiled to provide an integrative view on the molecular basis of CEP. The high-resolution structure of UROIIIS sheds light on the enzyme reaction mechanism while thermodynamic analysis revealed that the protein is thermolabile. Pathogenic missense mutations are found throughout the primary sequence of the enzyme. All but one of these is rarely found in patients, whereas C73R is responsible for more than one-third of the reported cases. Most of the mutant proteins (C73R included) retain partial catalytic activity but the mutations often reduce the enzyme's stability. The stabilization of the protein in vivo is discussed in the context of a new line of intervention to complement existing treatments such as bone marrow transplantation and gene therapy.
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Uroporphyrinogen III synthase mutations related to congenital erythropoietic porphyria identify a key helix for protein stability
Biochemistry, 2009Co-Authors: Arola Fortian, Ana Lain, David Castano, Gabriel Ortega, Miquel Pons, Oscar MilletAbstract:In the present study we have investigated deleterious mutants in the Uroporphyrinogen III synthase (UROIIIS) that are related to the congenital erythropoietic porphyria (CEP). The 25 missense mutan...
Heidi L. Schubert - One of the best experts on this subject based on the ideXlab platform.
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5 aminolaevulinic acid dehydratase porphobilinogen deaminase and Uroporphyrinogen III synthase
In: Warren MJ and Smith AG (eds.) Tetrapyrroles: Birth Life and Death. (pp. 43-73). Landes Bioscience: Austin. (2009), 2009Co-Authors: Heidi L. Schubert, P T Erskine, J B CooperAbstract:The three enzymes 5-aminolaevulinic acid dehydratase (ALAD, E.C.4.2.1.24; some times referred to as porphobilinogen synthase), porphobilinogen deaminase (EC 4.3.1.8; also known as hydroxymethylbilane synthase) and Uroporphyrinogen III synthase (U3S; E.C.4.2.1.75) together convert 5-aminolaevulinic acid (ALA) into Uroporphyrinogen III, from which all tetrapyrroles are synthesized. The X-ray structures of several ALADs have been determined showing that the enzyme forms a large homo-octameric structure with all eight active sites on the outer surface. Each subunit adopts the TIM barrel fold with an N-terminal arm which forms extensive inter-subunit interactions. The active site of each subunit is located in a pronounced cavity formed by loops at the C-terminal ends of the strands forming the TIM barrel. Current proposals for the catalytic mechanism involve the binding of both substrate moieties by formation of Schiff bases with two invariant active site lysine residues. Structural studies of porphobilinogen deaminase have shown that the enzyme has three domains, two of which show a strong structural resemblance to a number of periplasmic binding proteins. The reaction catalysed by Uroporphyrinogen III synthase involves cyclization and ring inversion, predicted to proceed through a spirocyclic intermediate. X-ray structures of the enzyme from humans and a thermophilic bacterium have enabled models of the catalytic process to be proposed.
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structure and mechanistic implications of a Uroporphyrinogen III synthase product complex
Biochemistry, 2008Co-Authors: Heidi L. Schubert, John D. Phillips, Annie Heroux, Christopher P. HillAbstract:Uroporphyrinogen III synthase (U3S) catalyzes the asymmetrical cyclization of a linear tetrapyrrole to form the physiologically relevant Uroporphyrinogen III (uro'gen III) isomer during heme biosynthesis. Here, we report four apoenzyme and one product complex crystal structures of the Thermus thermophilus (HB27) U3S protein. The overlay of eight crystallographically unique U3S molecules reveals a huge range of conformational flexibility, including a "closed" product complex. The product, uro'gen III, binds between the two domains and is held in place by a network of hydrogen bonds between the product's side chain carboxylates and the protein's main chain amides. Interactions of the product A and B ring carboxylate side chains with both structural domains of U3S appear to dictate the relative orientation of the domains in the closed enzyme conformation and likely remain intact during catalysis. The product C and D rings are less constrained in the structure, consistent with the conformational changes required for the catalytic cyclization with inversion of D ring orientation. A conserved tyrosine residue is potentially positioned to facilitate loss of a hydroxyl from the substrate to initiate the catalytic reaction.
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structure function studies on a s adenosyl l methionine dependent Uroporphyrinogen III c methyltransferase sumt a key regulatory enzyme of tetrapyrrole biosynthesis
Journal of Molecular Biology, 2004Co-Authors: J Vevodova, Heidi L. Schubert, Evelyne Raux, Ross M Graham, David I Roper, Amanda A Brindley, Ian A Scott, Charles A RoessnerAbstract:The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent Uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of Uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of Uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.
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Structural diversity in metal ion chelation and the structure of Uroporphyrinogen III synthase.
Biochemical Society Transactions, 2002Co-Authors: Heidi L. Schubert, Evelyne Raux, M. A. A. Matthews, John D. Phillips, Keith S. Wilson, Christopher P. HillAbstract:All tetrapyrroles are synthesized through a branched pathway, and although each tetrapyrrole receives unique modifications around the ring periphery, they all share the unifying feature of a central metal ion. Each pathway maintains a unique metal ion chelatase, and several tertiary structures have been determined, including those of the protoporphyrin ferrochelatase from both human and Bacillus subtilus , and the cobalt chelatase CbiK. These enzymes exhibit strong structural similarity and appear to function by a similar mechanism. Met8p, from Saccharomyces cerevisiae , catalyses ferrochelation during the synthesis of sirohaem, and the structure reveals a novel chelatase architecture whereby both ferrochelation and NAD + -dependent dehydrogenation take place in a single bifunctional active site. Asp-141 appears to participate in both catalytic reactions. The final common biosynthetic step in tetrapyrrole biosynthesis is the generation of Uroporphyrinogen by Uroporphyrinogen III synthase, whereby the D ring of hydroxymethylbilane is flipped during ring closure to generate the asymmetrical structure of Uroporphyrinogen III. The recently derived structure of Uroporphyrinogen III synthase reveals a bi-lobed structure in which the active site lies between the domains.
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crystal structure of human Uroporphyrinogen III synthase
The EMBO Journal, 2001Co-Authors: Michael A A Mathews, Heidi L. Schubert, John D. Phillips, Frank G Whitby, Kelly J Alexander, Kevin Schadick, Hector A Bergonia, Christopher P. HillAbstract:Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F 430 and vitamin B 12 . U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 A resolution. The protein folds into two α/β domains connected by a β‐ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.
Gunhild Layer - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the heme d1 biosynthesis enzyme nire in complex with its substrate reveals new insights into the catalytic mechanism of s adenosyl l methionine dependent Uroporphyrinogen III methyltransferases
Journal of Biological Chemistry, 2011Co-Authors: Sonja Storbeck, S Saha, Joern Krausze, B U Klink, Dirk W Heinz, Gunhild LayerAbstract:Abstract During the biosynthesis of heme d1, the essential cofactor of cytochrome cd1 nitrite reductase, the NirE protein catalyzes the methylation of Uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate Uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 A resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent Uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of Uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.
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the pseudomonas aeruginosa nire gene encodes the s adenosyl l methionine dependent Uroporphyrinogen III methyltransferase required for heme d1 biosynthesis
FEBS Journal, 2009Co-Authors: Sonja Storbeck, Johannes Walther, Judith Müller, Vina Parmar, Hans Martin Schiebel, Dorit Kemken, Thomas Dülcks, Gunhild LayerAbstract:Biosynthesis of heme d1, the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd1, requires the methylation of the tetrapyrrole precursor Uroporphyrinogen III at positions C-2 and C-7. We produced Pseudomonas aeruginosa NirE, a putative S-adenosyl-l-methionine (SAM)-dependent Uroporphyrinogen III methyltransferase, as a recombinant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography. Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer. NirE was shown to be a SAM-dependent Uroporphyrinogen III methyltransferase that catalyzes the conversion of Uroporphyrinogen III into precorrin-2 in vivo and in vitro. A specific activity of 316.8 nmol of precorrin-2 h−1·mg−1 of NirE was found for the conversion of Uroporphyrinogen III to precorrin-2. At high enzyme concentrations NirE catalyzed an overmethylation of Uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin. Substrate inhibition was observed at Uroporphyrinogen III concentrations above 17 μm. The protein did bind SAM, although not with the same avidity as reported for other SAM-dependent Uroporphyrinogen III methyltransferases involved in siroheme and cobalamin biosynthesis. A P. aeruginosa nirE transposon mutant was not complemented by native cobA encoding the SAM-dependent Uroporphyrinogen III methyltransferase involved in cobalamin formation. However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special requirement of NirE for heme d1 biosynthesis.
Kenneth H. Astrin - One of the best experts on this subject based on the ideXlab platform.
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congenital erythropoietic porphyria identification and expression of eight novel mutations in the Uroporphyrinogen III synthase gene
British Journal of Haematology, 2002Co-Authors: Amr A Shady, Luis F Cunha, Kenneth H. Astrin, Brandon R Colby, David F BishopAbstract:Summary. Mutations in the Uroporphyrinogen III synthase (URO-synthase) gene cause congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of haem biosynthesis. Molecular analysis of the URO-synthase gene in seven unrelated CEP patients revealed eight novel mutations. These included four missense mutations (A69T, E81D, G188W and I219S), a deletion (21delG), two insertions (398insG and 672ins28) and one complex mutation (627del6ins39), as well as three previously reported mutations, C73R, T228M, and −86CA. When the four novel missense mutations were expressed in Escherichia coli, only E81D expressed significant enzymatic activity (30% of expressed wild-type activity), which was thermolabile. In addition, reverse transcription polymerase chain reaction studies demonstrated that E81D, which altered the penultimate nucleotide in exon 4, impaired splicing and caused about 85% exon 4 skipping. The identification and expression of these mutations provided genotype–phenotype correlations and further evidence of the molecular heterogeneity underlying this erythropoietic porphyria.
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Uroporphyrinogen III synthase erythroid promoter mutations in adjacent gata1 and cp2 elements cause congenital erythropoietic porphyria
Journal of Clinical Investigation, 2001Co-Authors: Constanza Solis, Gerardo I. Aizencang, Kenneth H. Astrin, David F BishopAbstract:Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of Uroporphyrinogen III synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined allele identified four novel mutations clustered in a 20-bp region: (a) a -70T to C transition in a putative GATA-1 consensus binding element, (b) a -76G to A transition, (c) a -86C to A transversion in three unrelated patients, and (d) a -90C to A transversion in a putative CP2 binding motif. Also, a -224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 +/- 1, 54 +/- 3, 43 +/- 6, and 8 +/- 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophoretic mobility shift assays indicated that the -70C mutation altered GATA1 binding, whereas the adjacent -76A mutation did not. Similarly, the -90C mutation altered CP2 binding, whereas the -86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.
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Uroporphyrinogen III synthase an alternative promoter controls erythroid specific expression in the murine gene
Journal of Biological Chemistry, 2000Co-Authors: Gerardo I. Aizencang, David F Bishop, Douglas Forrest, Kenneth H. AstrinAbstract:Uroporphyrinogen III synthase (URO-synthase, EC 4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-synthase, the cDNA and approximately 24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5'-untranslated regions (5'-UTRs), but identical coding exons 2B through 10. Transcripts with 5'-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5'-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (<5% of wild type). Thus, housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-synthase gene.
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molecular basis of congenital erythropoietic porphyria mutations in the human Uroporphyrinogen III synthase gene
Human Mutation, 1996Co-Authors: Kenneth H. AstrinAbstract:Congenital erythropoietic porphyria (CEP) is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of the fourth enzyme in the heme biosynthetic pathway, Uroporphyrinogen III synthase (URO-synthase). To date, 17 mutations have been described including 11 missense, one nonsense, two mRNA splicing defects, one deletion and two coding region insertions. Most mutations have been identified in one or a few unrelated families with the exception of C73R and L4F which occurred in 29.6% and 9.3% of the 54 mutant alleles studied, respectively. Interestingly, analysis of the mutant alleles identified only 83% of the causative mutations, suggesting that about 20% of the mutations causing CEP lie elsewhere in the gene. Of note, mutation V82F, resulting from a G to T transversion of the last nucleotide of exon 4, caused both a missense mutation and an aberrantly spliced RNA transcript. Prokaryotic expression of the mutant URO-synthase alleles identified those with significant residual activity, thereby permitting genotype/phenotype predictions for this clinically heterogeneous disease.
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Regional assignment of the human Uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2→q26.3
Human Genetics, 1991Co-Authors: Kenneth H. Astrin, Hanwook Yoo, Cecilia A Warner, Paul J. Goodfellow, Shihfeng TsaiAbstract:Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%–53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2→ q26.3.
Charles A Roessner - One of the best experts on this subject based on the ideXlab platform.
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structure function studies on a s adenosyl l methionine dependent Uroporphyrinogen III c methyltransferase sumt a key regulatory enzyme of tetrapyrrole biosynthesis
Journal of Molecular Biology, 2004Co-Authors: J Vevodova, Heidi L. Schubert, Evelyne Raux, Ross M Graham, David I Roper, Amanda A Brindley, Ian A Scott, Charles A RoessnerAbstract:The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent Uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of Uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of Uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.
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Mutagenesis identifies a conserved tyrosine residue important for the activity of Uroporphyrinogen III synthase from Anacystis nidulans.
FEBS letters, 2002Co-Authors: Charles A Roessner, Krishan Ponnamperuma, A I ScottAbstract:Uroporphyrinogen III synthase from the cyanobacterium Anacystis nidulans was overproduced in Escherichia coli and analyzed by site specific mutagenesis. Of the nine conserved amino acids altered, only a single tyrosine mutant (Y166F) showed any significant decrease in activity suggesting this residue is critical for proper substrate binding and/or catalysis.
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cloning sequencing and expression of the Uroporphyrinogen III methyltransferase coba gene of propionibacterium freudenreichii shermanii
Journal of Bacteriology, 1995Co-Authors: I Sattler, Charles A Roessner, Neal J. Stolowich, Susan H Hardin, Larry W Harrishaller, N T Yokubaitis, Y Murooka, Y Hashimoto, A I ScottAbstract:We cloned, sequenced, and overexpressed cobA, the gene encoding Uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to Uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to Uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.