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Guangfu Yang - One of the best experts on this subject based on the ideXlab platform.
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synthesis in vitro Protoporphyrinogen oxidase inhibition and herbicidal activity of n benzothiazol 5 yl hexahydro 1h isoindole 1 3 diones and n benzothiazol 5 yl hexahydro 1h isoindol 1 ones
Chemical Biology & Drug Design, 2014Co-Authors: Lili Jiang, Yang Zuo, Zhifang Wang, Guangfu YangAbstract:Protoporphyrinogen oxidase (EC 1.3.3.4) is one of the most significant targets for a large family of herbicides. As part of our continuous efforts to search for novel Protoporphyrinogen oxidase-inhibiting herbicides, N-(benzothiazol-5-yl)tetrahydroisoindole-1,3-dione was selected as a lead compound for structural optimization, leading to the syntheses of a series of novel N-(benzothiazol-5-yl)hexahydro-1H-isoindole-1,3-diones (1a-o) and N-(benzothiazol-5-yl)hexahydro-1H-isoindol-1-ones (2a-i). These newly prepared compounds were characterized by elemental analyses, (1) H NMR, and ESI-MS, and the structures of 1h and 2h were further confirmed by X-ray diffraction analyses. The bioassays indicated that some compounds displayed comparable or higher Protoporphyrinogen oxidase inhibition activities in comparison with the commercial control. Very promising, compound 2a, ethyl 2-((6-fluoro-5-(4,5,6,7-tetrahydro-1-oxo-1H-isoindol-2(3H)-yl)benzo[d]thiazol-2-yl)-sulfanyl)acetate, was recognized as the most potent candidate with K(i) value of 0.0091 μm. Further greenhouse screening results demonstrated that some compounds exhibited good herbicidal activity against Chenopodium album at the dosage of 150 g/ha.
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computational and experimental insights into the mechanism of substrate recognition and feedback inhibition of Protoporphyrinogen oxidase
PLOS ONE, 2013Co-Authors: Gefei Hao, Zhifang Wang, Ying Tan, Changguo Zhan, Sheng-gang Yang, Guangfu YangAbstract:Protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) is an essential enzyme catalyzing the last common step in the pathway leading to heme and chlorophyll biosynthesis. Great interest in PPO inhibitors arises from both its significance to agriculture and medicine. However, the discovery of PPO inhibitors with ultrahigh potency and selectivity is hampered due to lack of structural and mechanistic understanding about the substrate recognition, which remains a longstanding question central in porphyrin biology. To understand the mechanism, a novel binding model of protogen (Protoporphyrinogen IX, the substrate) was developed through extensive computational simulations. Subsequently, amino acid residues that are critical for protogen binding identified by computational simulations were substituted by mutagenesis. Kinetic analyses of these mutants indicated that these residues were critical for protogen binding. In addition, the calculated free energies of protogen binding with these mutants correlated well with the experimental data, indicating the reasonability of the binding model. On the basis of this novel model, the fundamental mechanism of substrate recognition was investigated by performing potential of mean force (PMF) calculations, which provided an atomic level description of conformational changes and pathway intermediates. The free energy profile revealed a feedback inhibition mechanism of proto (protoporphyrin IX, the product), which was also in agreement with experimental evidence. The novel mechanistic insights obtained from this study present a new starting point for future rational design of more efficient PPO inhibitors based on the product-bound PPO structure.
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structural insight into human variegate porphyria disease
The FASEB Journal, 2011Co-Authors: Xiaohong Qin, Zhifang Wang, Guangfu Yang, Ying Tan, Xin Wen, Baifan Wang, Lele Wang, Yuequan ShenAbstract:Human Protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein, converts Protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway. Mutations in the hPPO gene cause the inherited human disease variegate porphyria (VP). In this study, we report the crystal structure of hPPO in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of 1.9 A. The structural and biochemical analyses revealed the molecular details of FAD and acifluorfen binding to hPPO as well as the interactions of the substrate with hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro. The founder-effect mutation R59W in VP patients is most likely caused by a severe electrostatic hindrance in the hydrophilic binding pocket involving the bulky, hydrophobic indolyl ring of the tryptophan. Forty-seven VP-causing mutations were purified by chromatography and kinetically characterized in vitro. The...
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understanding the mechanism of drug resistance due to a codon deletion in Protoporphyrinogen oxidase through computational modeling
Journal of Physical Chemistry B, 2009Co-Authors: Gefei Hao, Guangfu Yang, Xiaolei Zhu, Li Zhang, Changguo ZhanAbstract:Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the last common enzyme for the enzymatic transformation of Protoporphyrinogen-IX to protoporphyrin-IX, which is the key common intermediate leading to heme and chlorophyll. Hence, PPO has been identified as one of the most importance action targets for the treatment of some important diseases including cancer and variegated porphyria (VP). In the agricultural field, PPO inhibitors have been used as herbicides for many years. Recently, a unique drug resistance was found to be associated with a nonactive site residue (Gly210) deletion rather than substitution in A. tuberculatus PPO. In the present study, extensive computational simulations, including homology modeling, molecular dynamics (MD) simulations, and molecular mechanics-Poisson−Boltzmann surface area (MM-PBSA) calculations, have been carried out to uncover the detailed molecular mechanism of drug resistance associated with Gly210 deletion. Although Gly210 in the wild-type A. tuberculatus PPO has no di...
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A capillary electrophoresis assay for recombinant Bacillus subtilis Protoporphyrinogen oxidase.
Analytical biochemistry, 2008Co-Authors: Ying Tan, Guangfu Yang, Lu Sun, Dong Qing Jiang, Xiu-ping Yan, Xing YangAbstract:Abstract Protoporphyrinogen oxidase (PPO) is a flavin adenine dinucleotide (FAD)-containing enzyme in the tetrapyrrole biosynthetic pathway that leads to the formation of both heme and chlorophylls, which has been identified as one of the most important action targets of commercial herbicides. The literature reports gave different PPO-catalytic kinetic parameters for the substrate Protoporphyrinogen IX ( K m of 0.1 to 10.4 μM) with different sources of PPO using fluorescent or HPLC methods. Herein we assayed the enzymatic activity of recombinant Bacillus subtilis PPO by using capillary electrophoresis (CE), a method with high separation efficiency, easy automation, and low sample consumption. The Michaelis constant and maximum reaction velocity were determined as 7.0 ± 0.6 μM and 0.38 ± 0.02 μmol min -1 μg −1 , respectively. The interaction between PPO and acifluorfen, a commercial PPO-inhibiting herbicide, was measured as the inhibition constant 186.9 ± 9.3 μМ. The relationship between cofactor FAD and PPO activity can also be quantitatively studied by this CE method. The CE method used here should also be a convenient, reliable method for PPO study.
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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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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variegate porphyria in south africa 1688 1996 new developments in an old disease
South African Medical Journal, 1997Co-Authors: R J Hift, Harry A. Dailey, P.n. Meissner, Anne V. Corrigall, Mel Ziman, Doreen M Meissner, Brandon P Davidson, Lavinia A Petersen, Jean Sutherland, R. E. KirschAbstract:Variegate porphyria, an autosomal dominant inherited trait resulting in decreased activity of Protoporphyrinogen oxidase, the penultimate haem biosynthetic enzyme, is characterised clinically by photosensitive skin disease and a propensity to acute neurovisceral crises. The disease has an exceptionally high frequency in South Africa, owing to a founder effect. The specific mutation in the Protoporphyrinogen oxidase gene sequence which represents this founder gene has been identified. Genetic diagnosis is therefore now possible in families in whom the gene defect is known. However, the exact nature and degree of activity of the porphyria can only be determined by detailed quantitative biochemical analysis of excreted porphyrins. The relative contributions of the acute attack and the skin disease to the total disease burden of patients with variegate porphyria is not static, and in South Africa there have been significant changes over the past 25 years, with fewer patients presenting with acute attacks, leaving a greater proportion to present with skin disease or to remain asymptomatic with the diagnosis being made in the laboratory. The most common precipitating cause of the acute attack of VP is administration of porphyrinogenic drugs. Specific suppression of haem synthesis with intravenous haem arginate is the most useful treatment of a moderate or severe acute attack. Although cutaneous lesions are limited to the sun-exposed areas, management of the skin disease of VP remains inadequate.
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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
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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variegate porphyria past present and future
Skin Pharmacology and Applied Skin Physiology, 1998Co-Authors: Jorge Frank, Angela M ChristianoAbstract:Variegate porphyria, one of the acute hepatic porphyrias, is characterized by a partial reduction in Protoporphyrinogen oxidase, the seventh enzyme of the heme biosynthetic pathway. For a long time, this disease has caused confusion among the porphyrias because it presents with clinical symptoms and biochemical findings that can be similar to those found in other types of porphyrias. Here, we provide an overview of historical, clinical, biochemical, genetical, and other aspects of variegate porphyria that might be helpful in providing more insight into this rare disorder.
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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.
Zhifang Wang - One of the best experts on this subject based on the ideXlab platform.
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Computational and Experimental Insights into the Mechanism of Substrate Recognition and Feedback Inhibition of
2016Co-Authors: Protoporphyrinogen Oxidase, Zhifang Wang, Ying Tan, Gefei Hao, Sheng-gang Yang, Changguo ZhanAbstract:Protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) is an essential enzyme catalyzing the last common step in the pathway leading to heme and chlorophyll biosynthesis. Great interest in PPO inhibitors arises from both its significance to agriculture and medicine. However, the discovery of PPO inhibitors with ultrahigh potency and selectivity is hampered due to lack of structural and mechanistic understanding about the substrate recognition, which remains a longstanding question central in porphyrin biology. To understand the mechanism, a novel binding model of protogen (Protoporphyrinogen IX, the substrate) was developed through extensive computational simulations. Subsequently, amino acid residues that are critical for protogen binding identified by computational simulations were substituted by mutagenesis. Kinetic analyses of these mutants indicated that these residues were critical for protogen binding. In addition, the calculated free energies of protogen binding with these mutants correlated well with the experimental data, indicating the reasonability of the binding model. On the basis of this novel model, the fundamental mechanism of substrate recognition was investigated by performing potential of mean force (PMF) calculations, which provided an atomic level description of conformational changes and pathway intermediates. The free energy profile revealed a feedback inhibition mechanism of proto (protoporphyrin IX, the product), which was also in agreement with experimental evidence. The novel mechanistic insights obtained from this study present a new starting point for future rational design of more efficient PPO inhibitors based o
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synthesis in vitro Protoporphyrinogen oxidase inhibition and herbicidal activity of n benzothiazol 5 yl hexahydro 1h isoindole 1 3 diones and n benzothiazol 5 yl hexahydro 1h isoindol 1 ones
Chemical Biology & Drug Design, 2014Co-Authors: Lili Jiang, Yang Zuo, Zhifang Wang, Guangfu YangAbstract:Protoporphyrinogen oxidase (EC 1.3.3.4) is one of the most significant targets for a large family of herbicides. As part of our continuous efforts to search for novel Protoporphyrinogen oxidase-inhibiting herbicides, N-(benzothiazol-5-yl)tetrahydroisoindole-1,3-dione was selected as a lead compound for structural optimization, leading to the syntheses of a series of novel N-(benzothiazol-5-yl)hexahydro-1H-isoindole-1,3-diones (1a-o) and N-(benzothiazol-5-yl)hexahydro-1H-isoindol-1-ones (2a-i). These newly prepared compounds were characterized by elemental analyses, (1) H NMR, and ESI-MS, and the structures of 1h and 2h were further confirmed by X-ray diffraction analyses. The bioassays indicated that some compounds displayed comparable or higher Protoporphyrinogen oxidase inhibition activities in comparison with the commercial control. Very promising, compound 2a, ethyl 2-((6-fluoro-5-(4,5,6,7-tetrahydro-1-oxo-1H-isoindol-2(3H)-yl)benzo[d]thiazol-2-yl)-sulfanyl)acetate, was recognized as the most potent candidate with K(i) value of 0.0091 μm. Further greenhouse screening results demonstrated that some compounds exhibited good herbicidal activity against Chenopodium album at the dosage of 150 g/ha.
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computational and experimental insights into the mechanism of substrate recognition and feedback inhibition of Protoporphyrinogen oxidase
PLOS ONE, 2013Co-Authors: Gefei Hao, Zhifang Wang, Ying Tan, Changguo Zhan, Sheng-gang Yang, Guangfu YangAbstract:Protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) is an essential enzyme catalyzing the last common step in the pathway leading to heme and chlorophyll biosynthesis. Great interest in PPO inhibitors arises from both its significance to agriculture and medicine. However, the discovery of PPO inhibitors with ultrahigh potency and selectivity is hampered due to lack of structural and mechanistic understanding about the substrate recognition, which remains a longstanding question central in porphyrin biology. To understand the mechanism, a novel binding model of protogen (Protoporphyrinogen IX, the substrate) was developed through extensive computational simulations. Subsequently, amino acid residues that are critical for protogen binding identified by computational simulations were substituted by mutagenesis. Kinetic analyses of these mutants indicated that these residues were critical for protogen binding. In addition, the calculated free energies of protogen binding with these mutants correlated well with the experimental data, indicating the reasonability of the binding model. On the basis of this novel model, the fundamental mechanism of substrate recognition was investigated by performing potential of mean force (PMF) calculations, which provided an atomic level description of conformational changes and pathway intermediates. The free energy profile revealed a feedback inhibition mechanism of proto (protoporphyrin IX, the product), which was also in agreement with experimental evidence. The novel mechanistic insights obtained from this study present a new starting point for future rational design of more efficient PPO inhibitors based on the product-bound PPO structure.
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structural insight into human variegate porphyria disease
The FASEB Journal, 2011Co-Authors: Xiaohong Qin, Zhifang Wang, Guangfu Yang, Ying Tan, Xin Wen, Baifan Wang, Lele Wang, Yuequan ShenAbstract:Human Protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein, converts Protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway. Mutations in the hPPO gene cause the inherited human disease variegate porphyria (VP). In this study, we report the crystal structure of hPPO in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of 1.9 A. The structural and biochemical analyses revealed the molecular details of FAD and acifluorfen binding to hPPO as well as the interactions of the substrate with hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro. The founder-effect mutation R59W in VP patients is most likely caused by a severe electrostatic hindrance in the hydrophilic binding pocket involving the bulky, hydrophobic indolyl ring of the tryptophan. Forty-seven VP-causing mutations were purified by chromatography and kinetically characterized in vitro. The...
Lili Jiang - One of the best experts on this subject based on the ideXlab platform.
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synthesis in vitro Protoporphyrinogen oxidase inhibition and herbicidal activity of n benzothiazol 5 yl hexahydro 1h isoindole 1 3 diones and n benzothiazol 5 yl hexahydro 1h isoindol 1 ones
Chemical Biology & Drug Design, 2014Co-Authors: Lili Jiang, Yang Zuo, Zhifang Wang, Guangfu YangAbstract:Protoporphyrinogen oxidase (EC 1.3.3.4) is one of the most significant targets for a large family of herbicides. As part of our continuous efforts to search for novel Protoporphyrinogen oxidase-inhibiting herbicides, N-(benzothiazol-5-yl)tetrahydroisoindole-1,3-dione was selected as a lead compound for structural optimization, leading to the syntheses of a series of novel N-(benzothiazol-5-yl)hexahydro-1H-isoindole-1,3-diones (1a-o) and N-(benzothiazol-5-yl)hexahydro-1H-isoindol-1-ones (2a-i). These newly prepared compounds were characterized by elemental analyses, (1) H NMR, and ESI-MS, and the structures of 1h and 2h were further confirmed by X-ray diffraction analyses. The bioassays indicated that some compounds displayed comparable or higher Protoporphyrinogen oxidase inhibition activities in comparison with the commercial control. Very promising, compound 2a, ethyl 2-((6-fluoro-5-(4,5,6,7-tetrahydro-1-oxo-1H-isoindol-2(3H)-yl)benzo[d]thiazol-2-yl)-sulfanyl)acetate, was recognized as the most potent candidate with K(i) value of 0.0091 μm. Further greenhouse screening results demonstrated that some compounds exhibited good herbicidal activity against Chenopodium album at the dosage of 150 g/ha.
