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Peter M Shoolinginjordan - One of the best experts on this subject based on the ideXlab platform.
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crystallization and preliminary x ray characterization of the tetrapyrrole biosynthetic enzyme Porphobilinogen Deaminase from arabidopsis thaliana
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2012Co-Authors: A Roberts, Stephen P. Wood, P T Erskine, J B Cooper, R Gill, R J Hussey, Halina Mikolajek, E J T Chrystal, Peter M ShoolinginjordanAbstract:The enzyme Porphobilinogen Deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole Porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution.
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structure of human Porphobilinogen Deaminase at 2 8 a the molecular basis of acute intermittent porphyria
Biochemical Journal, 2009Co-Authors: R Gill, Simon E. Kolstoe, Fiyaz Mohammed, J B Cooper, S P Wood, M Sarwar, Abeer Al Dbass, Julie E Mosely, Peter M ShoolinginjordanAbstract:Mutations in the human PBGD (Porphobilinogen Deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P21212 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix α23 and forms a β-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed.
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human Porphobilinogen Deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation
Biochemical Society Transactions, 2003Co-Authors: Peter M Shoolinginjordan, A Aldbass, L A Mcneill, M Sarwar, D ButlerAbstract:Porphobilinogen Deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo-protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo-protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo-protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo-protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo-enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme–substrate intermediates from the catalytic cycle.
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acute intermittent porphyria in sweden molecular functional and clinical consequences of some new mutations found in the Porphobilinogen Deaminase gene
Clinical Genetics, 2002Co-Authors: Ylva Floderus, Peter M Shoolinginjordan, P HarperAbstract:Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial deficit of Porphobilinogen Deaminase (PBGD), the third of eight enzymes in the haem biosynthetic pathway. The overt disease is characterized by neuropsychiatric symptoms that are often triggered by exogenous factors such as certain drugs, stress, and alcohol. The aim of this work has been to identify the underlying genetic defect in each AIP-affected family in order to provide early counselling to assist in the avoidance of precipitating factors. The prevalence of AIP in Sweden is in the order of 1:10 000. The major mutation in Sweden, W198X, is due to a founder effect in the northern part of the country. This mutation, together with a further 11 mutations, have been reported previously. The present communication encompasses the great majority of AIP kindreds in Sweden and includes a further 27 mutations within the PBGD gene. This includes 14 completely new mutations, as well as 11 known mutations detected for the first time in Sweden. The majority of the mutations are located in exons 10 and 12 with fewer in exon 7. The clinical and biochemical outcomes in some patients are described. We also use the three-dimensional structure of the Porphobilinogen Deaminase enzyme to predict the possible molecular and functional consequences of the new Swedish missense and nonsense mutations.
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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.
Raili Kauppinen - One of the best experts on this subject based on the ideXlab platform.
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Acute Intermittent Porphyria: Expression of Mutant and Wild-Type Porphobilinogen Deaminase in COS-1 Cells
Molecular Medicine, 2000Co-Authors: Sami Mustajoki, Pertti Mustajoki, Minna Laine, Maija Lahtela, Leena Peltonen, Raili KauppinenAbstract:Background Acute intermittent porphyria (AIP) is an autosomal dominant disorder that results from the partial deficiency of Porphobilinogen Deaminase (PBGD) in the heme biosynthetic pathway. Patients with AIP can experience acute attacks consisting of abdominal pain and various neuropsychiatric symptoms. Although molecular biological studies on the Porphobilinogen Deaminase (PBGD) gene have revealed several mutations responsible for AIP, the properties of mutant PBGD in eukaryotic expression systems have not been studied previously. Materials and Methods Seven mutations were analyzed using transient expression of the mutated polypeptides in COS-1 cells. The properties of mutated polypeptides were studied by enzyme activity measurement, Western blot analysis, pulse-chase experiments, and immunofluorescence staining. Results Of the mutants studied, R26C, R167W, R173W, R173Q, and R225X resulted in a decreased enzyme activity (0–5%), but R225G and 1073delA (elongated protein) displayed a significant residual activity of 16% and 50%, respectively. In Western blot analysis, the polyclonal PBGD antibody detected all mutant polypeptides except R225X, which was predicted to result in a truncated protein. In the pulse-chase experiment, the mutant polypeptides were as stable as the wild-type enzyme. In the immunofluorescence staining both wild-type and mutant polypeptides were diffusely dispersed in the cytoplasm and, thus, no accumulation of mutated proteins in the cellular compartments could be observed. Conclusions The results confirm the causality of mutations for the half normal enzyme activity measured in the patients′ erythrocytes. In contrast to the decreased enzyme activity, the majority of the mutations produced a detectable polypeptide, and the stability and the intracellular processing of the mutated polypeptides were both comparable to that of the wild-type PBGD and independent of the cross-reacting immunological material (CRIM) class.
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acute intermittent porphyria expression of mutant and wild type Porphobilinogen Deaminase in cos 1 cells
Molecular Medicine, 2000Co-Authors: Sami Mustajoki, Pertti Mustajoki, Minna Laine, Maija Lahtela, Leena Peltonen, Raili KauppinenAbstract:Acute intermittent porphyria (AIP) is an autosomal dominant disorder that results from the partial deficiency of Porphobilinogen Deaminase (PBGD) in the heme biosynthetic pathway. Patients with AIP can experience acute attacks consisting of abdominal pain and various neuropsychiatric symptoms. Although molecular biological studies on the Porphobilinogen Deaminase (PBGD) gene have revealed several mutations responsible for AIP, the properties of mutant PBGD in eukaryotic expression systems have not been studied previously. Seven mutations were analyzed using transient expression of the mutated polypeptides in COS-1 cells. The properties of mutated polypeptides were studied by enzyme activity measurement, Western blot analysis, pulse-chase experiments, and immunofluorescence staining. Of the mutants studied, R26C, R167W, R173W, R173Q, and R225X resulted in a decreased enzyme activity (0–5%), but R225G and 1073delA (elongated protein) displayed a significant residual activity of 16% and 50%, respectively. In Western blot analysis, the polyclonal PBGD antibody detected all mutant polypeptides except R225X, which was predicted to result in a truncated protein. In the pulse-chase experiment, the mutant polypeptides were as stable as the wild-type enzyme. In the immunofluorescence staining both wild-type and mutant polypeptides were diffusely dispersed in the cytoplasm and, thus, no accumulation of mutated proteins in the cellular compartments could be observed. The results confirm the causality of mutations for the half normal enzyme activity measured in the patients′ erythrocytes. In contrast to the decreased enzyme activity, the majority of the mutations produced a detectable polypeptide, and the stability and the intracellular processing of the mutated polypeptides were both comparable to that of the wild-type PBGD and independent of the cross-reacting immunological material (CRIM) class.
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acute intermittent porphyria in finland 19 mutations in the Porphobilinogen Deaminase gene
Human Molecular Genetics, 1995Co-Authors: Raili Kauppinen, L Peltonen, S Mustajoki, H Pihlaja, Pertti MustajokiAbstract:The sensitivity of single-strand conformation polymorphism (SSCP) analysis for the detection of mutations in the Porphobilinogen Deaminase (PBGD) gene among Finnish patients with acute intermittent porphyria (AIP) was studied. 13 novel mutations including one de novo event, and six previously characterized mutations were identified among AIP patients. The 19 mutations reported here for 28 families cover 72% of all the AIP families in the Finnish population of five million. When compared to direct sequencing, SSCP-analysis detected 17 (89%) of the 19 mutations when a combination of various electrophoretic conditions were used. The most informative electrophoretic condition was a gel run without glycerol in the coldroom (11/18 mutations). 86% of mutations were identified from amplified fragments greater than 300 bp and detection was dependent on both the amount of glycerol in the gel and the running temperature, but seemed to be independent of the size of the analyzed fragment or the type of mutation. The diagnostic efficiency of biochemical assays versus mutation screening in the PBGD gene was studied in three large AIP families, each representing different CRIM subtypes of AIP. The results demonstrated that using assays of erythrocyte PBGD activity, the majority (82%) of family members (n = 51) were diagnosed correctly. Of a total of 81 family members, 30 of whom had deficiency of PBGD confined to non-erythroid tissues, diagnosis at the asymptomatic stage of disease in 11 individuals (14%) required the application of mutation screening.
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frequency of low erythrocyte Porphobilinogen Deaminase activity in finland
Journal of Internal Medicine, 1992Co-Authors: Pertti Mustajoki, Raili Kauppinen, L Lannfelt, L Lilius, J KoistinenAbstract:The frequency of low erythrocyte Porphobilinogen Deaminase (PBGD) activity was investigated in 2234 blood donors and in 30 patients with acute intermittent porphyria. The mean enzyme activities (+/- SD) were 3.38 +/- 0.58 U and 1.82 +/- 0.41 U, respectively. Eighteen blood donors without any history of symptoms of porphyria or haematological disease had low PBGD activity (less than 2.20 U), and they were studied further. All of them also had subnormal concentrations of the erythrocyte enzyme protein, as determined by an immunological method. Lymphocyte PBGD activity was within the normal range, but this parameter does exhibit a wide overlap between normal and porphyric values. Urinary excretion of Porphobilinogen was moderately increased in two of the blood donors. In four of the 18 families of the blood donors with low PBGD activity several first-degree relatives had low erythrocyte enzyme activity, consistent with a dominant mode of inheritance. The 5-aminolaevulinic acid loading-test was normal in the blood donors with familial occurrence of low erythrocyte PBGD. It is concluded that inherited defects in erythrocyte PBGD occurred among Finnish blood donors with a frequency of about 1 in 500. The defects may be identical with those in acute intermittent porphyria (AIP), but other mechanisms are also possible, e.g. a mutation in the erythroid-specific part of the PBGD gene.
Bernard Grandchamp - One of the best experts on this subject based on the ideXlab platform.
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Porphobilinogen Deaminase deficiency in mice causes a neuropathy resembling that of human hepatic porphyria
Nature Genetics, 1996Co-Authors: Raija L P Lindberg, Bernard Grandchamp, Catherine Porcher, Birgit Ledermann, Kurt Burki, Sebastian Brandner, Adriano Aguzzi, Urs A MeyerAbstract:Acute intermittent porphyria (AIP) is a human disease resulting from a dominantly inherited partial deficiency of the heme biosynthetic enzyme, Porphobilinogen Deaminase (PBGD). The frequency of the trait for AIP is 1/10,000 in most populations, but may be markedly higher (1/500) in psychiatric patients. The clinical expression of the disease is characterized by acute, life-threatening attacks of 'porphyric neuropathy' that include abdominal pain, motor and sensory neurological deficits and psychiatric symptoms. Attacks are frequently precipitated by drugs, alcohol and low caloric intake. Identical symptoms occur in other hepatic porphyrias. To study the pathogenesis of the neurologic symptoms of AIP we have generated Pbgd-deficient mice by gene targeting. These mice exhibit the typical biochemical characteristics of human AIP, notably, decreased hepatic Pbgd activity, increased delta-aminolevulinic acid synthase activity and massively increased urinary excretion of the heme precursor, delta-aminolevulinic acid after treatment with drugs such as phenobarbital. Behavioural tests reveal decreased motor function and histopathological findings include axonal neuropathy and neurologic muscle atrophy.
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High prevalence of a point mutation in the Porphobilinogen Deaminase gene in Dutch patients with acute intermittent porphyria
Human Genetics, 1993Co-Authors: Felix Rooij, Yves Nordmann, Jean-charles Deybach, J. S. Lee, Kor Velde, Bernard GrandchampAbstract:Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by a deficiency of Porphobilinogen Deaminase (PBGD). Up to now 14 different mutations have been described. In an effort to investigate the molecular epidemiology of AIP we have undertaken a systematic study of different exons of the PBGD gene from a large number of unrelated patients. Here, exon 8 from 82 unrelated Dutch and French AIP patients was examined using single strand confirmation polymorphism analysis (SSCP) after polymerase chain reaction (PCR) amplification. A single base mutation, C to T, at position 346 of the sequence coding for PBGD was observed in 15 Dutch families but in only 1 French family. A simple PCR assay is described to facilitate the diagnosis of this common mutation at the DNA level.
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association between genetic variation at the Porphobilinogen Deaminase gene and schizophrenia
Schizophrenia Research, 1993Co-Authors: Alan R Sanders, Bernard Grandchamp, Diego E Rinconlimas, Ranajit Chakraborty, Joseph D Hamilton, William E Fann, Pragna PatelAbstract:There is growing evidence that some genetic predisposition is important in the etiology of schizophrenia. We have sought to implicate a major gene by performing a candidate gene association study comparing the allele frequencies of seven restriction fragment length polymorphisms (RFLPs) at six loci in both a psychiatrically normal control group (N = 51) and an affected (schizophrenia or schizoaffective disorder) group (N = 55). Each group comprised Caucasians of northern European origin. The candidate areas (D5S39, D5S78, dopamine receptor D2 (DRD2), D11S29, Porphobilinogen Deaminase (PBGD), and D11S84) were selected on the basis of prior cytogenetic findings in schizophrenics, linkage studies, and/or implicated gene products. The presence of a polymorphic ApaLI site within the PBGD gene showed a significant association with the presence of illness (P = 0.02). The relative risk of possessing the allele with the ApaLI site was 2.10. No significant association was found with any of the six other RFLPs. Our data suggests that either the PBGD gene itself or an unknown gene linked to and/or in linkage disequilibrium with the PBGD locus predisposes some individuals to schizophrenia. Independent replication of these findings will be required to determine their relevance to schizophrenia.
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denaturing gradient gel electrophoresis for rapid detection of latent carriers of a subtype of acute intermittent porphyria with normal erythrocyte Porphobilinogen Deaminase activity
Clinical Chemistry, 1992Co-Authors: F Bourgeois, Y Nordmann, J C Deybach, M Te P Velde, F W M De Rooij, Bernard GrandchampAbstract:Acute intermittent porphyria is an autosomal dominant disorder defined by a partial deficiency of Porphobilinogen Deaminase (EC 4.3.1.8). Clinical manifestations of the disease are characterized by acute attacks of neurological dysfunction often linked to environmental factors. Early diagnosis of gene carriers is important in the prevention of attacks and is usually achieved by determining the Porphobilinogen Deaminase activity in erythrocytes. However, in a subtype of acute intermittent porphyria, the enzymatic defect is restricted to nonerythropoietic cells. Different mutations have already been described that account for this phenotype in two unrelated families. We previously detected asymptomatic carriers by using mutation-specific probes after in vitro amplification of the target DNA sequence. In this study, we investigated the DNA of eight unrelated subjects with the same subtype of acute intermittent porphyria by using the polymerase chain reaction, with subsequent analysis of the amplified products by denaturing gradient gel electrophoresis. Five of these patients shared the same single-base change. This technique was quite simple and efficient for detecting asymptomatic carriers. Importantly, it is potentially useful for studying families with the same phenotypic subtype of the disease and possibly different mutations in the same DNA region.
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characterization of hypersensitive sites protein binding motifs and regulatory elements in both promoters of the mouse Porphobilinogen Deaminase gene
Journal of Biological Chemistry, 1991Co-Authors: C Porcher, G Pitiot, M Plumb, S Lowe, H De Verneuil, Bernard GrandchampAbstract:Porphobilinogen Deaminase, the third enzyme in the heme biosynthetic pathway, is encoded by a gene having two different promoters. Differential splicing of transcripts from the promoters yields two distinct mRNA species that are translated to give two isoforms of the protein. One isoform is ubiquitous, whereas the other is erythroid-specific. In this study, we have analyzed the gene regulatory elements that contribute to the tissue-specific promoter utilization of the mouse Porphobilinogen Deaminase gene. Six nuclear DNase I-hypersensitive sites were mapped in erythroid and nonerythroid cells, and four of these regions were further analyzed for in vitro nuclear protein-binding sites. The erythroid-specific promoter contains three erythroid nuclear factor GF-1-binding sites. The proximal GF-1-binding site, together with an adjacent duplicated CACCC motif, was sufficient to confer erythroid-specific expression in functional studies. Furthermore, as upstream gene sequences were shown to greatly increase promoter activity in erythroid cells, it suggests an upstream erythroid-specific enhancer may also be required for the up-regulation of the erythroid-specific promoter during erythropoiesis.
Peter M Jordan - One of the best experts on this subject based on the ideXlab platform.
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subcellular location of the tetrapyrrole synthesis enzyme Porphobilinogen Deaminase in higher plants an immunological investigation
Planta, 1996Co-Authors: Michael Witty, Peter M Jordan, Russell M Jones, Matthew S Robb, Alison G SmithAbstract:A recombinant plasmid, pArab8, harbouring the cDNA encoding the mature form of the tetrapyrrole synthesis enzyme Porphobilinogen Deaminase (EC 4.3.1.8; also known as hydroxymethylbilane synthase) from Arabidopsis thaliana (L.) Heynh. has been constructed, and used to transform Escherichia coli. The Porphobilinogen Deaminase protein from Arabidopsis was overexpressed in this strain, and purified to homogeneity (3000-fold) with a yield of 20%. Antibodies were raised against the purified plant enzyme, and used in Western blot analysis, immunoprecipitation of enzyme activity and immuno-gold electron microscopy. The results indicate that the enzyme is confined to plastids in both leaves and roots. The implications of this finding for plant tetrapyrrole synthesis are discussed.
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the three dimensional structures of mutants of Porphobilinogen Deaminase toward an understanding of the structural basis of acute intermittent porphyria
Protein Science, 1994Co-Authors: Paul D Brownlie, Peter M Jordan, Jon B Cooper, Steve P Wood, Richard Lambert, Tom L Blundell, Gordon V Louie, Martin J WarrenAbstract:Mutations in the human gene for the enzyme Porphobilinogen Deaminase give rise to an inherited disease of heme biosynthesis, acute intermittent porphyria. Knowledge of the 3-dimensional structure of human Porphobilinogen Deaminase, based on the structure of the bacterial enzyme, allows correlation of structure with gene organization and leads to an understanding of the relationship between mutations in the gene, structural and functional changes of the enzyme, and the symptoms of the disease. Most mutations occur in exons 10 and 12, often changing amino acids in the active site. Several of these are shown to be involved in binding the primer or substrate; none modifies Asp 84, which is essential for catalytic activity.
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purification and properties of Porphobilinogen Deaminase from arabidopsis thaliana
Biochemical Journal, 1994Co-Authors: R M Jones, Peter M JordanAbstract:Porphobilinogen Deaminase (EC 4.3.1.8) has been purified to homogeneity (16,000-fold) from the plant Arabidopsis thaliana in yields of 8%. The Deaminase is a monomer of M(r) 35,000, as shown by SDS/PAGE, and 31,000, using gel-filtration chromatography. The pure enzyme has a Vmax. of 4.5 mumol/h per mg and a Km of 17 +/- 4 microM. Determination of the pI and pH optimum revealed values of 5.2 and 8.0 respectively. The sequence of the N-terminus was found to be NH2-XVAVEQKTRTAI. The Deaminase is heat-stable up to 70 degrees C and is inhibited by NH3 and hydroxylamine. The enzyme is inactivated by arginine-, histidine- and lysine-specific reagents. Incubation with the substrate analogue and suicide inhibitor, 2-bromoPorphobilinogen, results in chain termination and in inactivation.
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structure of Porphobilinogen Deaminase reveals a flexible multidomain polymerase with a single catalytic site
Nature, 1992Co-Authors: Gordon V Louie, Martin J Warren, J B Cooper, Paul D Brownlie, Richard Lambert, Tom L Blundell, S P Wood, Sarah C Woodcock, Peter M JordanAbstract:The three-domain structure of Porphobilinogen Deaminase, a key enzyme in the biosynthetic pathway of tetrapyrroles, has been defined by X-ray analysis at 1.9 A resolution. Two of the domains structurally resemble the transferrins and periplasmic binding proteins. The dipyrromethane cofactor is covalently linked to domain 3 but is bound by extensive salt-bridges and hydrogen-bonds within the cleft between domains 1 and 2, at a position corresponding to the binding sites for small-molecule ligands in the analogous proteins. The X-ray structure and results from site-directed mutagenesis provide evidence for a single catalytic site. Interdomain flexibility may aid elongation of the polypyrrole product in the active-site cleft of the enzyme.
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mutagenesis of arginine residues in the catalytic cleft of escherichia coli Porphobilinogen Deaminase that affects dipyrromethane cofactor assembly and tetrapyrrole chain initiation and elongation
Biochemical Journal, 1991Co-Authors: Peter M Jordan, Sarah C WoodcockAbstract:Substitutions of conserved arginine residues in the catalytic cleft of Escherichia coli Porphobilinogen Deaminase were constructed by site-specific mutagenesis of the hemC gene. Mutant proteins exhibited a range of defects including the failure to assemble the dipyrromethane cofactor and the inability to initiate and propagate the tetrapolymerization reaction. Mutations of arginine residues at positions 11, 131, 132 and 155, all of which interact with the carboxylic acid side chains of the dipyrromethane cofactor, were the most disruptive.
J B Cooper - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of pyrrole polymerization in human Porphobilinogen Deaminase
2018Co-Authors: P Pluta, J B Cooper, P Roversi, G Bernardo-seisdedos, Al Rojas, Rw Pickersgill, O MilletAbstract:Human Porphobilinogen Deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert Porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria
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structural studies of domain movement in active site mutants of Porphobilinogen Deaminase from bacillus megaterium
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2017Co-Authors: Jingxu Guo, P T Erskine, J B Cooper, S P Wood, Alun R CokerAbstract:The enzyme Porphobilinogen Deaminase (PBGD) is one of the key enzymes in tetrapyrrole biosynthesis. It catalyses the formation of a linear tetrapyrrole from four molecules of the substrate Porphobilinogen (PBG). It has a dipyrromethane cofactor (DPM) in the active site which is covalently linked to a conserved cysteine residue through a thioether bridge. The substrate molecules are linked to the cofactor in a stepwise head-to-tail manner during the reaction, which is catalysed by a conserved aspartate residue: Asp82 in the B. megaterium enzyme. Three mutations have been made affecting Asp82 (D82A, D82E and D82N) and their crystal structures have been determined at resolutions of 2.7, 1.8 and 1.9 A, respectively. These structures reveal that whilst the D82E mutant possesses the DPM cofactor, in the D82N and D82A mutants the cofactor is likely to be missing, incompletely assembled or disordered. Comparison of the mutant PBGD structures with that of the wild-type enzyme shows that there are significant domain movements and suggests that the enzyme adopts `open' and `closed' conformations, potentially in response to substrate binding.
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crystallization and preliminary x ray characterization of the tetrapyrrole biosynthetic enzyme Porphobilinogen Deaminase from bacillus megaterium
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2013Co-Authors: N Azim, Martin J Warren, Stephen P. Wood, P T Erskine, J B Cooper, Evelyne Deery, M AkhtarAbstract:The enzyme Porphobilinogen Deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole Porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution.
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crystallization and preliminary x ray characterization of the tetrapyrrole biosynthetic enzyme Porphobilinogen Deaminase from arabidopsis thaliana
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2012Co-Authors: A Roberts, Stephen P. Wood, P T Erskine, J B Cooper, R Gill, R J Hussey, Halina Mikolajek, E J T Chrystal, Peter M ShoolinginjordanAbstract:The enzyme Porphobilinogen Deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole Porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution.
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structure of human Porphobilinogen Deaminase at 2 8 a the molecular basis of acute intermittent porphyria
Biochemical Journal, 2009Co-Authors: R Gill, Simon E. Kolstoe, Fiyaz Mohammed, J B Cooper, S P Wood, M Sarwar, Abeer Al Dbass, Julie E Mosely, Peter M ShoolinginjordanAbstract:Mutations in the human PBGD (Porphobilinogen Deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P21212 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix α23 and forms a β-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed.