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Gunhild Layer - One of the best experts on this subject based on the ideXlab platform.
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Tetrapyrrolbiosynthese in denitrifizierenden Bakterien und Archaea
BIOspektrum, 2011Co-Authors: Sonja Storbeck, Gunhild LayerAbstract:The biosynthesis of heme d _1 in denitrifying bacteria and heme in the Archaea proceeds via the methylated intermediate precorrin-2. The involved Uroporphyrinogen III methyltransferases utilize arginine as catalytically active base. Die Biosynthese von Häm d _1 in denitrifizierenden Bakterien und Häm in Archaea verläuft ungewübnlicherweise über die methylierte Vorstufe Precorrin-2. Die beteiligten Uroporphyrinogen-III-Methyltransferasen nutzen Arginin als katalytisch aktive Base.
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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, Martin J. Warren, 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.
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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, Martin J. Warren, 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.
Alan R. Battersby - One of the best experts on this subject based on the ideXlab platform.
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expression purification and characterisation of the product from the bacillus subtilis hemd gene Uroporphyrinogen iii synthase
FEBS Journal, 1995Co-Authors: Patrick N J Stamford, Alfredo Capretta, Alan R. BattersbyAbstract:Uroporphyrinogen III synthase, the product of the hemD gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane. The hemD gene isolated from Bacillus subtilis was manipulated by PCR to enable direct cloning behind a synthetic ribosome-binding site downstream of tandem bacteriophage λPR and PL promoters in a pCE30-derived vector. Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of Uroporphyrinogen III synthase. The protein produced was soluble and was readily purified. Pure Uroporphyrinogen III synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3. Its specific activity by assay using synthetic hydroxymethylbilane as substrate is 565 units mg−1 and the Km for this substrate is 330 ± 30 nM. The N -terminal sequence of the enzyme is Met-Glu-Asn-Asp-Phe-Pro-Leu, in agreement with the gene-derived sequence. Studies based on amino acid modifications suggest that arginine, lysine and probably histidine residues are essential for the activity of Uroporphyrinogen III synthase. Significantly, this synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.
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Uroporphyrinogen III synthase: Studies on its mechanism of action, molecular biology and biochemistry
Tetrahedron, 1991Co-Authors: Nigel Crockett, Peter R. Alefounder, Alan R. Battersby, Chris AbellAbstract:Abstract A review is given of studies on the mechanism of action of Uroporphyrinogen III synthase (cosynthetase, EC 4.2.1.75) based on synthesis of an inhibitory spiro-lactam. HemD, the Escherichia coli gene coding for Uroporphyrinogen III synthase has been cloned and overexpressed at levels sixteen fold higher than in wild type E. coli. Evidence for hemD being part of an operon is reviewed. Uroporphyrinogen III synthase, which has been purified approx. 6000 fold, shows Mr 28000 under denaturing conditions and has a pH optimum of 8.0 ± 0.2. The results from chemical modification of the enzyme point to the presence of arginine and lysine residues at or close to the active site.
Peter M Shoolinginjordan - One of the best experts on this subject based on the ideXlab platform.
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porphobilinogen deaminase and Uroporphyrinogen iii synthase structure molecular biology and mechanism
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Peter M ShoolinginjordanAbstract:Porphobilinogen deaminase (hydroxymethylbilane synthase) and Uroporphyrinogen III synthase (Uroporphyrinogen III cosynthase) catalyze the transformation of four molecules of porphobilinogen, via the 1-hydroxymethylbilane, preUroporphyrinogen, into Uroporphyrinogen III. A combination of studies involving protein chemistry, molecular biology, site-directed mutagenesis, and the use of chemically synthesized substrate analogs and inhibitors is helping to unravel the complex mechanisms by which the two enzymes function. The determination of the X-ray structure ofE. coli porphobilinogen deaminase at 1.76 A resolution has provided the springboard for the design of further experiments to elucidate the precise mechanism for the assembly of both the dipyrromethane cofactor and the tetrapyrrole chain. The human deaminase structure has been modeled from theE. coli structure and has led to a molecular explanation for the disease acute intermittent porphyria. Molecular modeling has also been employed to simulate the spiro-mechanism of Uroporphyrinogen III synthase.
Chang Kee Lim - One of the best experts on this subject based on the ideXlab platform.
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Porphomethene inhibitor of Uroporphyrinogen decarboxylase: analysis by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.
Biomedical Chromatography, 2007Co-Authors: Malcolm Danton, Chang Kee LimAbstract:An uroporphomethene inhibitor of Uroporphyrinogen decarboxylase, characterized by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry, was reported recently (Phillips et al., Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 5079-5084). Close examination of the tandem mass spectrometric fragmentation pattern of the compound showed that it is not a tetrapyrrole or an Uroporphyrinogen or uroporphyrin related molecule. The product ion spectrum showed a fragmentation pattern typical of a poly(ethylene glycol) structure. Characteristic fragmentations of the side-chain acetic acid and propionic acid substituents of a uroporphyrin or Uroporphyrinogen derivative were absent.
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porphomethene inhibitor of Uroporphyrinogen decarboxylase analysis by high performance liquid chromatography electrospray ionization tandem mass spectrometry
Biomedical Chromatography, 2007Co-Authors: Malcolm Danton, Chang Kee LimAbstract:An uroporphomethene inhibitor of Uroporphyrinogen decarboxylase, characterized by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry, was reported recently (Phillips et al., Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 5079-5084). Close examination of the tandem mass spectrometric fragmentation pattern of the compound showed that it is not a tetrapyrrole or an Uroporphyrinogen or uroporphyrin related molecule. The product ion spectrum showed a fragmentation pattern typical of a poly(ethylene glycol) structure. Characteristic fragmentations of the side-chain acetic acid and propionic acid substituents of a uroporphyrin or Uroporphyrinogen derivative were absent.
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Preparation and separation of hydroxy derivatives of Uroporphyrinogen I by high-performance liquid chromatography with electrochemical detection.
Journal of Chromatography A, 1996Co-Authors: Chang Kee LimAbstract:The preparation and high-performance liquid chromatography (HPLC) separation of meso-hydroxyUroporphyrinogen I, hydroxyacetic acid Uroporphyrinogen I and β-hydroxypropionic acid Uroporphyrinogen I is described. meso-Hydroxyuroporphyrin I, hydroxyacetic acid uroporphyrin I and β-hydroxypropionic acid uroporphyrin I were isolated from the urine of a patient with congenital erythropoietic porphyria. The porphyrins were reduced to the corresponding porphyrinogens with 3% (w/w) Na/Hg amalgam. The hydroxy porphyrinogens were separated on a Hypersil ODS column with 4% (v/v) acetonitrile in 1 M ammonium acetate buffer, pH 5.16, containing EDTA (0.27 mM) as the mobile phase, and detected electrochemically. Reduction of meso-hydroxyuroporphyrin I and hydroxyacetic acid uroporphyrin I, followed by HPLC analysis, showed that, in addition to the expected formation of meso-hydroxyUroporphyrinogen I and hydroxyacetic Uroporphyrinogen I, respectively, Uroporphyrinogen I was also produced. Reduction of β-hydroxypropionic acid uroporphyrin I, however, gave β-hydroxypropionic acid Uroporphyrinogen I, acrylic acid Uroporphyrinogen I and Uroporphyrinogen I as the products. The peaks were identified by conversion into the porphyrin methyl esters and analysed by liquid secondary-ion mass spectrometry.
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Order of Uroporphyrinogen III decarboxylation on incubation of porphobilinogen and Uroporphyrinogen III with erythrocyte Uroporphyrinogen decarboxylase.
The Biochemical journal, 1993Co-Authors: Jinli Luo, Chang Kee LimAbstract:The isomeric compositions of the heptacarboxylic, hexacarboxylic and pentacarboxylic porphyrinogens formed by incubation of porphobilinogen with human red-cell haemolysates have been analysed and compared with those derived from incubation with chemically prepared Uroporphyrinogen III as substrate. The results indicated that when supplied with an excess (3.7 microM) of exogenous Uroporphyrinogen III, Uroporphyrinogen decarboxylase utilized the substrate at random and a mixture of isomers was produced; whereas with Uroporphyrinogen III generated enzymically from porphobilinogen as substrate a clockwise decarboxylation sequence was observed, resulting in the formation of intermediates mainly with the ring-D, rings-AD and rings-ABD acetate groups decarboxylated. Using [14C]Uroporphyrinogen III as substrate at low concentrations (0.01-0.5 microM) also led to preferential decarboxylation of the ring-D acetate group. It was concluded that the order of Uroporphyrinogen III decarboxylation is substrate-concentration-dependent, and under normal physiological conditions enzymic decarboxylation is most probably orderly and clockwise, starting at the ring-D acetate group.
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Random decarboxylation of Uroporphyrinogen III by human hepatic Uroporphyrinogen decarboxylase.
Journal of Chromatography B: Biomedical Sciences and Applications, 1991Co-Authors: Jinli Luo, Chang Kee LimAbstract:The type III heptacarboxylic porphyrinogens derived from enzymic decarboxylation of an acetic acid substituent on Uroporphyrinogen III to a methyl group by human hepatic Uroporphyrinogen decarboxylase has been analysed by reversed-phase high-performance liquid chromatography with electrochemical detection. The results showed that all four possible heptacarboxylic acid porphyrinogen isomers, with the methyl group attached to rings A, B, C and D of the tetrapyrrole macrocycle, respectively, were formed in almost equal proportions. It was concluded that the normal pathway of Uroporphyrinogen III decarboxylation in human liver follows a random mechanism.
Sonja Storbeck - One of the best experts on this subject based on the ideXlab platform.
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Tetrapyrrolbiosynthese in denitrifizierenden Bakterien und Archaea
BIOspektrum, 2011Co-Authors: Sonja Storbeck, Gunhild LayerAbstract:The biosynthesis of heme d _1 in denitrifying bacteria and heme in the Archaea proceeds via the methylated intermediate precorrin-2. The involved Uroporphyrinogen III methyltransferases utilize arginine as catalytically active base. Die Biosynthese von Häm d _1 in denitrifizierenden Bakterien und Häm in Archaea verläuft ungewübnlicherweise über die methylierte Vorstufe Precorrin-2. Die beteiligten Uroporphyrinogen-III-Methyltransferasen nutzen Arginin als katalytisch aktive Base.
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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, Martin J. Warren, 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.
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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, Martin J. Warren, 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.
