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Jean-michel Camadro - One of the best experts on this subject based on the ideXlab platform.
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Acylation stabilizes a protease-resistant conformation of Protoporphyrinogen Oxidase, the molecular target of diphenyl ether-type herbicides.
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Sylvain Arnould, Masayuki Takahashi, Jean-michel CamadroAbstract:Protein acylation is an important way in which a number of proteins with a variety of functions are modified. The physiological role of the acylation of cellular proteins is still poorly understood. Covalent binding of fatty acids to nonintegral membrane proteins is thought to produce transient or permanent enhancement of the association of the polypeptide chains with biological membranes. In this paper, we investigate the functional role for the palmitoylation of an atypical membrane-bound protein, yeast Protoporphyrinogen Oxidase, which is the molecular target of diphenyl ether-type herbicides. Palmitoylation stabilizes an active heat- and protease-resistant conformation of the protein. Palmitoylation of Protoporphyrinogen Oxidase has been demonstrated to occur in vivo both in yeast cells and in a heterologous bacterial expression system, where it may be inhibited by cerulenin leading to the accumulation of degradation products of the protein. The thiol ester linking palmitoleic acid to the polypeptide chain was shown to be sensitive to hydrolysis by hydroxylamine and also by the widely used serine-protease inhibitor phenylmethylsulfonyl fluoride.
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Functional analysis of the hemK gene product involvement in Protoporphyrinogen Oxidase activity in yeast.
FEMS microbiology letters, 1999Co-Authors: Laurence Le Guen, Renata Santos, Jean-michel CamadroAbstract:The Escherichia coli hemK gene has been described as being involved in Protoporphyrinogen Oxidase activity; however, there is no biochemical evidence for this. In the context of characterizing the mechanisms of Protoporphyrinogen oxidation in the yeast Saccharomyces cerevisiae, we investigated the yeast homolog of HemK, which is encoded by the ORF YNL063w, to find out whether it has any Protoporphyrinogen Oxidase activity and/or whether it modulates Protoporphyrinogen Oxidase activity. Phenotype analysis and enzyme activity measurements indicated that the yeast HemK homolog is not involved in Protoporphyrinogen Oxidase activity. Complementation assays in which the yeast HemK homolog is overproduced do not restore wild-type phenotypes in a yeast strain with deficient Protoporphyrinogen Oxidase activity. Protein sequence analysis of HemK-related proteins revealed consensus motif for S-adenosyl-methionine-dependent methyltransferase.
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Characteristics of Protoporphyrinogen Oxidase
Peroxidizing Herbicides, 1999Co-Authors: Jean-michel Camadro, Michel Matringe, Sylvain Arnould, Laurence Le Guen, Renata Santos, René MornetAbstract:Protoporphyrinogen Oxidase (EC 1.3.3.4) catalyzes the oxidative O2-dependent aromatization of the colorless Protoporphyrinogen IX to the highly conjugated protoporphyrin IX, the precursor of both hemes and chlorophylls (Fig. 1). It is the final enzyme in the common branch of the heme and chlorophyll biosyn-thetic pathways in plants (Fig. 2).
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Stability of recombinant yeast Protoporphyrinogen Oxidase: effects of diphenyl ether-type herbicides and diphenyleneiodonium.
Biochemistry, 1998Co-Authors: Sylvain Arnould, Masayuki Takahashi, Jean-michel CamadroAbstract:Protoporphyrinogen Oxidase catalyzes the oxygen-dependent aromatization of Protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides. Structural features of yeast Protoporphyrinogen Oxidase were assessed by circular dichroism studies on the enzyme purified from E. coli cells engineered to overproduce the protein. Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing Protoporphyrinogen Oxidase as a native, active, membrane-bound flavoprotein. The secondary structure of the Protoporphyrinogen Oxidase was 40.0 +/- 1. 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil. Purified Protoporphyrinogen Oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC). Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD). However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation. Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1. The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers. Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor. Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.
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The domain structure of Protoporphyrinogen Oxidase, the molecular target of diphenyl ether-type herbicides.
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Sylvain Arnould, Jean-michel CamadroAbstract:Protoporphyrinogen Oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast Protoporphyrinogen Oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for Protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of Protoporphyrinogen Oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of Protoporphyrinogen Oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.
Harry A. Dailey - One of the best experts on this subject based on the ideXlab platform.
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Discovery of a gene involved in a third bacterial Protoporphyrinogen Oxidase activity through comparative genomic analysis and functional complementation.
Applied and environmental microbiology, 2011Co-Authors: Tye O. Boynton, Svetlana Gerdes, Sarah H. Craven, Ellen L. Neidle, John D. Phillips, Harry A. DaileyAbstract:Tetrapyrroles are ubiquitous molecules in nearly all living organisms. Heme, an iron-containing tetrapyrrole, is widely distributed in nature, including most characterized aerobic and facultative bacteria. A large majority of bacteria that contain heme possess the ability to synthesize it. Despite this capability and the fact that the biosynthetic pathway has been well studied, enzymes catalyzing at least three steps have remained "missing" in many bacteria. In the current work, we have employed comparative genomics via the SEED genomic platform, coupled with experimental verification utilizing Acinetobacter baylyi ADP1, to identify one of the missing enzymes, a new Protoporphyrinogen Oxidase, the penultimate enzyme in heme biosynthesis. COG1981 was identified by genomic analysis as a candidate protein family for the missing enzyme in bacteria that lacked HemG or HemY, two known Protoporphyrinogen Oxidases. The predicted amino acid sequence of COG1981 is unlike those of the known enzymes HemG and HemY, but in some genomes, the gene encoding it is found neighboring other heme biosynthetic genes. When the COG1981 gene was deleted from the genome of A. baylyi, a bacterium that lacks both hemG and hemY, the organism became auxotrophic for heme. Cultures accumulated porphyrin intermediates, and crude cell extracts lacked Protoporphyrinogen Oxidase activity. The heme auxotrophy was rescued by the presence of a plasmid-borne Protoporphyrinogen Oxidase gene from a number of different organisms, such as hemG from Escherichia coli, hemY from Myxococcus xanthus, or the human gene for Protoporphyrinogen Oxidase.
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identification of escherichia coli hemg as a novel menadione dependent flavodoxin with Protoporphyrinogen Oxidase activity
Biochemistry, 2009Co-Authors: Tye O. Boynton, Tamara A. Dailey, Lauren E Daugherty, Harry A. DaileyAbstract:Protoporphyrinogen Oxidase (PPO, EC 1.3.3.4) catalyzes the six-electron oxidation of Protoporphyrinogen IX to the fully conjugated protoporphyrin IX. Eukaryotes and Gram-positive bacteria possess an oxygen-dependent, FAD-containing enzyme for this step, while the majority of Gram-negative bacteria lack this oxygen-dependent PPO. In Escherichia coli, PPO activity is known to be linked to respiration and the quinone pool. In E. coli SASX38, the knockout of hemG causes a loss of measurable PPO activity. HemG is a small soluble protein typical of long chain flavodoxins. Herein, purified recombinant HemG was shown to be capable of a menadione-dependent conversion of Protoporphyrinogen IX to protoporphyrin IX. Electrochemical analysis of HemG revealed similarities to other flavodoxins. Interestingly, HemG, a member of a class of the long chain flavodoxin family that is unique to the γ-proteobacteria, possesses a 22-residue sequence that, when transferred into E. coli flavodoxin A, produces a chimera that will c...
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PerOxidase activity of cytochrome c facilitates the Protoporphyrinogen Oxidase reaction
Cellular and molecular biology (Noisy-le-Grand France), 2009Co-Authors: Mark Shepherd, Harry A. DaileyAbstract:Protoporphyrinogen Oxidase (PPO) catalyzes the penultimate reaction in heme biosynthesis. The 'oxygen dependent' form of this enzyme can utilize three molecules of oxygen as electron acceptors in the reaction. In the current study, the ability of cytochrome c to serve as an electron acceptor for PPO was examined. Cytochrome c was found to enhance the catalytic rate of Drosophila melanogaster PPO under reduced oxygen conditions, and cytochrome c became reduced during PPO catalysis. Further kinetic analysis under anaerobic conditions revealed that hydrogen peroxide, a byproduct of the PPO reaction, is required for this rate enhancement to occur. This suggests that the generation of free radicals via the perOxidase activity of cytochrome c plays a part in this rate enhancement, rather than cytochrome c acting as an electron acceptor for the PPO reaction. Given the abundance of cytochrome c in the intermembrane space of mitochondria, the cellular location of PPO, this process may potentially impact on the synthesis of heme in vivo particularly in conditions of low oxygen or hypoxia.
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a continuous fluorimetric assay for Protoporphyrinogen Oxidase by monitoring porphyrin accumulation
Analytical Biochemistry, 2005Co-Authors: Mark Shepherd, Harry A. DaileyAbstract:Abstract A continuous spectrofluorimetric assay for Protoporphyrinogen Oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless nonfluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent K m ( K app ) values for Protoporphyrinogen IX were measured as 3.8 ± 0.3, 3.6 ± 0.5, and 1.0 ± 0.1 μM for the enzymes from human, Myxococcus xanthus , and Aquifex aeolicus , respectively. The K i for acifluorfen, a diphenylether herbicide, was measured as 0.53 μM for the human enzyme. Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h −1 /mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min −1 , representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.
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Characterization of the mouse Protoporphyrinogen Oxidase gene.
Cellular and molecular biology (Noisy-le-Grand France), 2002Co-Authors: Tamara A. Dailey, Julie F. Mcmanus, Harry A. DaileyAbstract:The murine Protoporphyrinogen Oxidase gene has been isolated, characterized and localized. The gene spans 4.2 kb, is comprised of 13 exons and 12 introns, and is located on chromosome 1 in band 1 H2. Analysis of 1.2 kb of the 5' upstream region revealed a promoter which is not GC rich and lacks any TATA boxes or initiator elements in the vicinity of the transcription start site. A variety of putative transcriptional element binding sequences were identified and gel shift assays support the presence of two GATA-1 sites near -760 bp as well as AP-1, AP-2, and Sp1 sites in the -1200 bp 5' flanking region. Luciferase reporter constructs transiently expressed in erythroid cell lines demonstrated erythroid-specific expression with the -1160 bp, but not with the -746 bp or -198 bp constructs. Expression in nonerythroid cells occurred maximally with -1160 bp, but was significant with -746 bp and absent with -198 bp. Expression of both housekeeping and erythroid-specific fusions in the transient expression systems was greatly decreased in the -5000 bp constructs suggesting the presence of repressor elements in the -1160 to -5000 bp region.
Angela M Christiano - One of the best experts on this subject based on the ideXlab platform.
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Identification of a founder mutation in the Protoporphyrinogen Oxidase gene in variegate porphyria patients from chile.
Human heredity, 2001Co-Authors: Jorge Frank, Vincent M. Aita, Wasim Ahmad, Hamut Lam, Wolff C, Angela M ChristianoAbstract:Variegate porphyria (VP; OMIM 176200) is characterized by a partial defect in the activity of Protoporphyrinogen Oxidase (PPO), the seventh enzyme of the porphyrin-heme biosynthetic pathway. The disea
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Mutations in the translation initiation codon of the Protoporphyrinogen Oxidase gene underlie variegate porphyria.
Clinical and experimental dermatology, 1999Co-Authors: Jorge Frank, Maureen B. Poh-fitzpatrick, John A. Mcgrath, John L.m. Hawk, Angela M ChristianoAbstract:Variegate porphyria (VP), one of the acute hepatic porphyrias, is characterized by a reduced catalytic activity of Protoporphyrinogen Oxidase (PPO), the penultimate enzyme in the porphyrin-haem biosynthetic pathway. VP has been linked to the PPO gene on chromosome 1q22-23, and several mutations underlying this disorder have been described recently. In this study, we identified two different missense mutations in the translation initiation codon of the PPO gene in two unrelated patients with VP. Mutation analysis was carried out using PCR, heteroduplex analysis, automated sequencing, and restriction enzyme digestion. In the first patient, the results revealed an A-to-T transversion (ATG --> TTG), resulting in the substitution of methionine by leucine (M1L). The mutation detected in the second patient was a T-to-C transition (ATG --> ACG), leading to the conversion of methionine to threonine (M1T). These mutations abolish the initiation of translation at the normal site, and consequently, translation of an abnormal messenger RNA (mRNA) would result in the synthesis of a truncated PPO protein lacking the amino terminus.
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Recurrent missense mutation in the Protoporphyrinogen Oxidase gene underlies variegate porphyria
American journal of medical genetics, 1998Co-Authors: Jorge Frank, Frank K. Jugert, Günter Goerz, Hans F. Merk, Claudia Breitkopf, Angela M ChristianoAbstract:The porphyrias represent a heterogeneous group of disorders of porphyrin or porphyrin-precursor metabolism, resulting from the inherited or acquired dysregulation of one of the eight enzymes in the porphyrin-heme biosynthetic pathway. Variegate porphyria, one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of the penultimate enzyme in the heme biosynthetic pathway, Protoporphyrinogen Oxidase (PPO). Recently, VP has been linked to the PPO gene on chromosome 1q22−23, and several disease-causing mutations have been described. In this study, we identified the underlying genetic lesion in two unrelated patients with VP and investigated all available family members by polymerase chain reaction, heteroduplex analysis, automated sequencing, and restriction enzyme digestion. Mutation analyses in both families revealed a G-to-A transition in exon 6 of the PPO gene resulting in the substitution of arginine by histidine at position 168 of the protein (R168H). This arginine residue is evolutionarily conserved in human, mouse, and Bacillus subtilis, indicating the importance of this residue in PPO function. Our study establishes a recurrent missense mutation as the underlying genetic defect in two unrelated patients with VP and explains the occurrence of the phenotype in their families. Am. J. Med. Genet. 79:22–26, 1998. © 1998 Wiley-Liss, Inc.
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Variegate Porphyria: Identification of a Nonsense Mutation in the Protoporphyrinogen Oxidase Gene
Journal of Investigative Dermatology, 1998Co-Authors: Jorge Frank, Frank K. Jugert, Katrin Kalka, Günter Goerz, Hans F. Merk, Angela M ChristianoAbstract:The porphyrias are disorders of porphyrin metabolism that result from inherited or acquired aberrations in the control of the heme biosynthetic pathway. Variegate porphyria is characterized by a partial reduction in the activity of Protoporphyrinogen Oxidase. In this study, we identified the first nonsense mutation in a family with variegate porphyria. The mutation consisted of a previously unreported G-to-T transversion in exon 5 of the Protoporphyrinogen Oxidase gene, resulting in the substitution of glutamic acid by a nonsense codon, designated E133X. Our investigation establishes that a nonsense mutation in the Protoporphyrinogen Oxidase gene is the underlying mutation in this family with variegate porphyria.
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homozygous variegate porphyria identification of mutations on both alleles of the Protoporphyrinogen Oxidase gene in a severely affected proband
Journal of Investigative Dermatology, 1998Co-Authors: Jorge Frank, John Mcgrath, Robert M Graham, J L M Hawk, Angela M ChristianoAbstract:Homozygous variegate porphyria is a severe skin and neurologic disease manifesting in early infancy, and characterized by markedly reduced levels of the penultimate enzyme in the heme biosynthetic pathway, Protoporphyrinogen Oxidase. We investigated the molecular basis of variegate porphyria, usually an autosomal dominantly inherited trait, in a severely affected female proband and her parents. The mutation detection strategy included heteroduplex analysis, automated sequencing, and allele specific oligonucleotide hybridization. We identified two underlying missense mutations in the Protoporphyrinogen Oxidase gene, consisting of a G-to-A transition in exon 6 (G169E), and a G-to-A transition in exon 10 (G358R). Our study establishes the molecular basis of “homozygous” variegate porphyria for the first time, in demonstrating that this patient is a compound heterozygote for two different missense mutations in the Protoporphyrinogen Oxidase gene.
P.n. Meissner - One of the best experts on this subject based on the ideXlab platform.
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The expression of Protoporphyrinogen Oxidase in human tissues.
Cellular and molecular biology (Noisy-le-Grand France), 2009Co-Authors: Anne V. Corrigall, K.b. Siziba, J A H Campbell, R. E. Kirsch, P.n. MeissnerAbstract:Protoporphyrinogen Oxidase is the penultimate enzyme in the haem biosynthetic pathway. In this study, the expression of Protoporphyrinogen Oxidase in a variety of human organs has been documented by immunohistochemical means at the light microscopy level in order to shed light on its inter- and intra-organ distribution. The expression varied amongst organs and the various cell types within an organ. The pattern of staining generally reflected presumed metabolic functionality and haem demand. Strongest staining was noted in hepatocytes, proximal convoluted tubules of the kidney, serous cells of the peribronchial gland in the lung, parietal cells of the stomach, tips of the villi in the small intestine and interstitial cells of the testis. Our results suggest that there are some significant sites of haem synthesis in addition to the liver and bone marrow, and should be borne in mind in studies related to haem or porphyrin dynamics and flux.
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Mitochondrial targeting of human Protoporphyrinogen Oxidase
Cell biology international, 2006Co-Authors: Lester M. Davids, Anne V. Corrigall, P.n. MeissnerAbstract:Abstract Variegate porphyria is an autosomal dominant disorder of heme metabolism resulting from a deficiency in Protoporphyrinogen Oxidase, an enzyme located on the inner mitochondrial membrane. This study examined the effect of three South African VP-causing mutations (H20P, R59W, R168C) on mitochondrial targeting. Only H20P did not target, and of eight Protoporphyrinogen Oxidase–GFP chimeric fusion proteins created, N-terminal residues 1–17 were found to be the minimal Protoporphyrinogen Oxidase sequence required for efficient mitochondrial targeting. Removal of this N-terminal sequence displayed mitochondrial localization, suggesting internal mitochondrial targeting signals. In addition, six constructs were engineered to assess the effect of charge and helicity on mitochondrial targeting of the protein. Of those engineered, only the PPOX20/H20P-GFP construct abolished mitochondrial targeting, presumably through disruption of the Protoporphyrinogen Oxidase α-helix. Based on our results we propose a mechanism for Protoporphyrinogen Oxidase targeting to the mitochondrion.
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Purification of and Kinetic Studies on a Cloned Protoporphyrinogen Oxidase from the Aerobic BacteriumBacillus subtilis
Archives of biochemistry and biophysics, 1998Co-Authors: Anne V. Corrigall, Ralph E. Kirsch, Tamara A. Dailey, Harry A. Dailey, K.b. Siziba, Mbulelo H. Maneli, E.g. Shephard, Melanie Ziman, P.n. MeissnerAbstract:Abstract The previously cloned and expressed Protoporphyrinogen Oxidase from Bacillus subtilis has been purified to homogeneity by Ni 2+ affinity chromatography using a His 6 tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a p I of 7.5, a pH optimum (Protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor. The Michaelis constants ( K m ) for Protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 μM, respectively. Polyclonal antibody to B. subtilis Protoporphyrinogen Oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus Protoporphyrinogen Oxidase. B. subtilis Protoporphyrinogen Oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 μM and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme.
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Identification and characterisation of a deletion (537delAT) in the Protoporphyrinogen Oxidase gene in a South African variegate porphyria family.
Human mutation, 1998Co-Authors: Anne V. Corrigall, Ralph E. Kirsch, Lester M. Davids, Doreen M Meissner, Richard J Hift, Valerie Hancock, P.n. MeissnerAbstract:Variegate porphyria is an autosomal dominant disorder of haem metabolism resulting from a partial decrease in Protoporphyrinogen Oxidase activity. Variegate porphyria is highly prevalent in South Africa, the result of a founder effect now confirmed genetically as a single point mutation (R59W) which has been described in nearly all South African variegate porphyria patients studied. Only two other mutations (H20P, R168C) have been reported in South Africa. We utilised simultaneous, single-stranded conformational polymorphism and heteroduplex analysis, and direct sequencing to identify a further mutation; a 2 bp deletion in exon 6 which results in a premature stop codon 11 codons downstream from the mutation and is the first reported deletion in the Protoporphyrinogen Oxidase gene in a South African family. The familial segregation of this mutation strongly suggests that it is the disease causing mutation for variegate porphyria in this family. This further evidence for allelic heterogeneity limits the utility of tests for the R59W mutation in the diagnosis of variegate porphyria in South Africa.
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a r59w mutation in human Protoporphyrinogen Oxidase results in decreased enzyme activity and is prevalent in south africans with variegate porphyria
Nature Genetics, 1996Co-Authors: P.n. Meissner, Anne V. Corrigall, Tamara A. Dailey, R. E. Kirsch, R J Hift, Mel Ziman, Andrew G Roberts, Doreen M Meissner, Harry A. DaileyAbstract:A R59W mutation in human Protoporphyrinogen Oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria
Kyoungwhan Back - One of the best experts on this subject based on the ideXlab platform.
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Use of Myxococcus xanthus Protoporphyrinogen Oxidase as a selectable marker for transformation of rice
Pesticide Biochemistry and Physiology, 2007Co-Authors: Kyung Jin Lee, Kiwoung Yang, Kiyoon Kang, Sei Kang, Nansook Lee, Kyoungwhan BackAbstract:Abstract Protoporphyrinogen Oxidase (PPO) is the target enzyme of peroxidizing herbicides. The overexpression of Myxococcus xanthus PPO ( Mx PPO) confers a high level of herbicide resistance in rice. Among the peroxidizing herbicides, butafenacil has an efficiency ∼1000-fold that of oxadiazon, as judged by calli susceptibility tests upon herbicide treatment. Butafenacil (0.1 μM) was used to select transgenic rice plants expressing Mx PPO under the control of the constitutive maize ubiquitin promoter. The ectopic expression of the Mx PPO transgene was investigated in the T 0 generation by Northern blot and Western blot analysis. The T 0 transgenic plants expressing the Mx PPO gene were resistant to butafenacil based on in vitro leaf disk and in vivo foliar spray tests.
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Expression of recombinant Protoporphyrinogen Oxidase influences growth and morphological characteristics in transgenic rice
Plant Growth Regulation, 2004Co-Authors: Sunyo Jung, Jung Sung Chung, Sang-uk Chon, Kyoungwhan BackAbstract:Transgenic rice plants expressing a Bacillus subtilis Protoporphyrinogen Oxidase (Protox), the last shared enzyme of the porphyrin pathway, in the cytoplasm (C89) or the plastids (P72) were compared with wild-type rice plants in their growth characteristics. Production of tiller buds 18 d after seeding was more profuse in transgenic plants than in wild-type plants, especially in plastid-targeted plants. Transgenic plants had 12–27% increase in tiller number and 17–33% increase in above-ground biomass compared with wild-type plants 4 and 8 weeks after transplanting of 2-week-old rice seedlings, demonstrating that tiller production and above-ground biomass correlate with each other. Cytoplasm-expressed and plastid-targeted transgenic plants also had a distinct phenotypic characteristic of narrower and more horizontal leaves than wild-type plants. Phenotypic and anatomical characteristics of the transgenic plants were clearly different from wild-type plants, indicating that regulation of porphyrin biosynthesis by expression of B. subtilis Protox in rice influences morphological characteristics of plant growth as well as biomass.
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Transgenic rice plants expressing Bacillus subtilis Protoporphyrinogen Oxidase gene show low herbicide oxyfluorfen resistance
Biologia Plantarum, 2003Co-Authors: S. B. Lee, Ja-ock Guh, D.e. Lee, Kyoungwhan BackAbstract:Transgenic rice plants harbouring Bacillus subtilis Protoporphyrinogen Oxidase (Protox) gene, which is targeted into plastid, were generated by Agrobacterium-mediated transformation using a rice (Oryza sativa L. cv. Dongjin) and their gene integration at T1 generation by Southern and mRNA expression in T2 generation by Northern blotting were analyzed. Their herbicide-resistant trait was further confirmed by in vitro leaf segment assay and in planta bioassays such as seed germination assay and measurement of growth inhibition. The herbicide oxyfluorfen resistance in transgenic rice plants was not very high. The results showed that the Protox from B. subtilis can not be applicable as a gene source to generate a high level oxyfluorfen tolerance in plants.
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A point mutation of valine-311 to methionine in Bacillus subtilis Protoporphyrinogen Oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen
Bioorganic chemistry, 2003Co-Authors: Eunjoo Jeong, Thavrak Houn, Yong In Kuk, Eun-seon Kim, Hema Kumar Chandru, Myunggi Baik, Kyoungwhan Back, Ja-ock Guh, Oksoo HanAbstract:In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis Protoporphyrinogen Oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis Protoporphyrinogen Oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant Protoporphyrinogen Oxidase was kinetically characterized. The mutant Protoporphyrinogen Oxidase showed very similar kinetic patterns to wild type Protoporphyrinogen Oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant Protoporphyrinogen Oxidase showed an increased inhibition constant about 1.5 times that of wild type Protoporphyrinogen Oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis Protoporphyrinogen Oxidase may not be an important determinant in the mechanism that protects Protoporphyrinogen Oxidase against diphenyl ether herbicides.
