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Tomoyoshi Nozaki - One of the best experts on this subject based on the ideXlab platform.
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discovery of antiamebic compounds that inhibit Cysteine Synthase from the enteric parasitic protist entamoeba histolytica by screening of microbial secondary metabolites
Frontiers in Cellular and Infection Microbiology, 2018Co-Authors: Mihoko Mori, Tomoyoshi Nozaki, Ghulam Jeelani, Kenichi Nonaka, Atsuko Matsumoto, Satoshi Tsuge, Wataru Fukasawa, Kumiko Nakadatsukui, Satoshi ōmura, Kazuro ShiomiAbstract:Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-Cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-Cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and Cysteine Synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo Cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the Cysteine-deprived medium compared to the Cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the Cysteine-deprived medium when compared to the growth in the Cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed Cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of Cysteine than that in the presence of Cysteine), although the IC50 value in the Cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of Cysteine. The Cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the Cysteine-containing medium, the Cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the Cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of Cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a Cysteine-dependent manner with relatively low mammalian cytotoxicity.
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discovery of antiamebic compounds that inhibit Cysteine Synthase from the enteric parasitic protist entamoeba histolytica by screening of microbial secondary metabolites
Frontiers in Cellular and Infection Microbiology, 2018Co-Authors: Mihoko Mori, Tomoyoshi Nozaki, Ghulam Jeelani, Kenichi Nonaka, Atsuko Matsumoto, Satoshi Tsuge, Wataru Fukasawa, Kumiko Nakadatsukui, Satoshi ōmura, Kazuro ShiomiAbstract:Amebiasis is caused by infection with the parasite, Entamoeba histolytica. Although metronidazole has been used against amebiasis, it shows side effects and low efficacy against asymptomatic cyst carriers. Therefore, drugs with new mode of action or targets are urgently needed. L-Cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica. E. histolytica possesses the de novo L-Cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and Cysteine Synthase (CS). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established the new screening system to discover antiamebic compounds that target the de novo Cysteine biosynthesis. The screening allowed us to identify the compounds that differentially affect the growth of the trophozoites in the Cysteine-deprived media compared to the Cysteine-containing medium. A total of 431 compounds of the Kitasato Natural Products Library and 6,900 of microbial culture broth extracts were screened. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in Cysteine-deprived medium when compared to the growth in the Cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural unstability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed Cysteine deprivation-dependent antiamebic activity, although the IC50 value in the Cysteine-deprived medium was rather high (283 M). Pencolide also showed inhibitory activity against EhCSs with comparable IC50 values. These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is attacked by Michael donors including the thiol moiety of Cysteine. The Cysteine-adducts were detected by mass spectrometric analysis as predicted. Thus, we cannot exclude a possibility that the Cysteine-adducts of pencolide may contribute Cysteine-dependent toxicity of pencolide to the parasite. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a Cysteine-dependent manner with relatively low mammalian cytotoxicity.
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Data_Sheet_1_Discovery of Antiamebic Compounds That Inhibit Cysteine Synthase From the Enteric Parasitic Protist Entamoeba histolytica by Screening of Microbial Secondary Metabolites.pdf
2018Co-Authors: Mihoko Mori, Tomoyoshi Nozaki, Ghulam Jeelani, Kenichi Nonaka, Atsuko Matsumoto, Satoshi Tsuge, Wataru Fukasawa, Kumiko Nakada-tsukui, Satoshi Ōmura, Kazuro ShiomiAbstract:Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-Cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-Cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and Cysteine Synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo Cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the Cysteine-deprived medium compared to the Cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the Cysteine-deprived medium when compared to the growth in the Cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed Cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of Cysteine than that in the presence of Cysteine), although the IC50 value in the Cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of Cysteine. The Cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the Cysteine-containing medium, the Cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the Cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of Cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a Cysteine-dependent manner with relatively low mammalian cytotoxicity.
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Genetic, metabolomic and transcriptomic analyses of the de novo L-Cysteine biosynthetic pathway in the enteric protozoan parasite Entamoeba histolytica
Nature Publishing Group, 2017Co-Authors: Ghulam Jeelani, Dan Sato, Tomoyoshi Soga, Tomoyoshi NozakiAbstract:Abstract The de novo L-Cysteine biosynthetic pathway is critical for the growth, antioxidative stress defenses, and pathogenesis of bacterial and protozoan pathogens, such as Salmonella typhimurium and Entamoeba histolytica. This pathway involves two key enzymes, serine acetyltransferase (SAT) and Cysteine Synthase (CS), which are absent in mammals and therefore represent rational drug targets. The human parasite E. histolytica possesses three SAT and CS isozymes; however, the specific roles of individual isoforms and significance of such apparent redundancy remains unclear. In the present study, we generated E. histolytica cell lines in which CS and SAT expression was knocked down by transcriptional gene silencing. The strain in which CS1, 2 and 3 were simultaneously silenced and the SAT3 gene-silenced strain showed impaired growth when cultured in a Cysteine lacking BI-S-33 medium, whereas silencing of SAT1 and SAT2 had no effects on growth. Combined transcriptomic and metabolomic analyses revealed that, CS and SAT3 are involved in S-methylCysteine/Cysteine synthesis. Furthermore, silencing of the CS1-3 or SAT3 caused upregulation of various iron-sulfur flavoprotein genes. Taken together, these results provide the first direct evidence of the biological importance of SAT3 and CS isoforms in E. histolytica and justify the exploitation of these enzymes as potential drug targets
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entamoeba histolytica phosphoserine aminotransferase ehpsat insights into the structure function relationship
BMC Research Notes, 2010Co-Authors: Vibhor Mishra, Tomoyoshi Nozaki, Vinod BhakuniAbstract:Background Presence of phosphorylated Serine biosynthesis pathway upstream to the de novo Cysteine biosynthesis pathway makes PSAT a crucial enzyme. Besides this, phoshoserine produced by the enzyme can also be taken up directly by Cysteine Synthase as a substrate. PSAT is a PLP dependent enzyme where the cofactor serves as an epicenter for functional catalysis with the active site architecture playing crucial role in optimum function of the enzyme.
Markus Wirtz - One of the best experts on this subject based on the ideXlab platform.
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the redox sensitive module of cyclophilin 20 3 2 Cysteine peroxiredoxin and Cysteine Synthase integrates sulfur metabolism and oxylipin signaling in the high light acclimation response
Plant Journal, 2017Co-Authors: Sara Mareike Muller, Markus Wirtz, Andrea Viehhauser, Shanshan Wang, Wilena Telman, Michael Liebthal, Helena Schnitzer, Carsten Sticht, Carolina Delatorre, Rudiger HellAbstract:Summary The integration of redox- and reactive oxygen species-dependent signaling and metabolic activities are fundamental to plant acclimation to biotic and abiotic stresses. Previous data suggest the existence of a dynamically interacting module in the chloroplast stroma consisting of cyclophilin 20-3 (Cyp20-3), O-acetylserine(thiol)lyase B (OASTL-B), 2-Cysteine peroxiredoxins A/B (2-CysPrx) and serine acetyltransferase 2;1 (SERAT2;1). The functionality of this COPS module is influenced by redox stimuli and oxophytodienoic acid (OPDA) which is the precursor for jasmonic acid. The concept of an integrating function of these proteins in stress signaling was challenged by combining transcriptome and biochemical analyses in Arabidopsis mutants devoid of oastlB, serat 2;1, cyp20-3 and 2-cysprxA/B, and wildtype. Leaf transcriptomes were analyzed 6h after transfer to light intensity 10-fold in excess of growth light or under growth light. The survey of KEGG-based gene ontology groups showed common upregulation of translation- and protein homeostasis-associated transcripts under control conditions in all mutants compared to wildtype. The results revealed that the interference of the module was accompanied with disturbance of carbohydrate, sulfur and nitrogen metabolism and also citric acid cycle intermediates. Apart from common regulation, specific responses at the transcriptome and metabolite level linked Cyp20-3 to cell wall-bound carbohydrates and oxylipin signaling, and 2-CysPrx to photosynthesis, sugar and amino acid metabolism. Deletion of either OASTL-B or SERAT2;1 frequently induced antagonistic changes in biochemical or molecular features. Enhanced sensitivity of mutant seedlings to OPDA and leaf discs to NaHS-administration confirmed the presumed functional interference of the COPS module in redox and oxylipin signaling. This article is protected by copyright. All rights reserved.
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the redox sensitive module of cyclophilin 20 3 2 Cysteine peroxiredoxin and Cysteine Synthase integrates sulfur metabolism and oxylipin signaling in the high light acclimation response
Plant Journal, 2017Co-Authors: Sara Mareike Muller, Markus Wirtz, Andrea Viehhauser, Shanshan Wang, Wilena Telman, Michael Liebthal, Helena Schnitzer, Carsten Sticht, Carolina Delatorre, Rudiger HellAbstract:The integration of redox- and reactive oxygen species-dependent signaling and metabolic activities is fundamental to plant acclimation to biotic and abiotic stresses. Previous data suggest the existence of a dynamically interacting module in the chloroplast stroma consisting of cyclophilin 20-3 (Cyp20-3), O-acetylserine(thiol)lyase B (OASTL-B), 2-Cysteine peroxiredoxins A/B (2-CysPrx) and serine acetyltransferase 2;1 (SERAT2;1). The functionality of this COPS module is influenced by redox stimuli and oxophytodienoic acid (OPDA), which is the precursor for jasmonic acid. The concept of an integrating function of these proteins in stress signaling was challenged by combining transcriptome and biochemical analyses in Arabidopsis mutants devoid of oastlB, serat2;1, cyp20-3 and 2-cysprxA/B, and wild-type (WT). Leaf transcriptomes were analyzed 6 h after transfer to light intensity 10-fold in excess of growth light or under growth light. The survey of KEGG-based gene ontology groups showed common upregulation of translation- and protein homeostasis-associated transcripts under control conditions in all mutants compared with WT. The results revealed that the interference of the module was accompanied with disturbance of carbohydrate, sulfur and nitrogen metabolism, and also citric acid cycle intermediates. Apart from common regulation, specific responses at the transcriptome and metabolite level linked Cyp20-3 to cell wall-bound carbohydrates and oxylipin signaling, and 2-CysPrx to photosynthesis, sugar and amino acid metabolism. Deletion of either OASTL-B or SERAT2;1 frequently induced antagonistic changes in biochemical or molecular features. Enhanced sensitivity of mutant seedlings to OPDA and leaf discs to NaHS-administration confirmed the presumed functional interference of the COPS module in redox and oxylipin signaling.
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molecular biology biochemistry and cellular physiology of Cysteine metabolism in arabidopsis thaliana
The Arabidopsis Book, 2011Co-Authors: Rudiger Hell, Markus WirtzAbstract:Cysteine is one of the most versatile molecules in biology, taking over such different functions as catalysis, structure, regulation and electron transport during evolution. Research on Arabidopsis has contributed decisively to the understanding of Cysteine synthesis and its role in the assimilatory pathways of S, N and C in plants. The multimeric Cysteine Synthase complex is present in the cytosol, plastids and mitochondria and forms the centre of a unique metabolic sensing and signaling system. Its association is reversible, rendering the first enzyme of Cysteine synthesis active and the second one inactive, and vice-versa. Complex formation is triggered by the reaction intermediates of Cysteine synthesis in response to supply and demand and gives rise to regulation of genes of sulfur metabolism to adjust cellular sulfur homeostasis. Combinations of biochemistry, forward and reverse genetics, structural- and cell-biology approaches using Arabidopsis have revealed new enzyme functions and the unique pattern of spatial distribution of Cysteine metabolism in plant cells. These findings place the synthesis of Cysteine in the centre of the network of primary metabolism.
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dominant negative modification reveals the regulatory function of the multimeric Cysteine Synthase protein complex in transgenic tobacco
The Plant Cell, 2007Co-Authors: Markus Wirtz, Rudiger HellAbstract:Cys synthesis in plants constitutes the entry of reduced sulfur from assimilatory sulfate reduction into metabolism. The catalyzing enzymes serine acetyltransferase (SAT) and O-acetylserine (OAS) thiol lyase (OAS-TL) reversibly form the heterooligomeric Cys Synthase complex (CSC). Dominant-negative mutation of the CSC showed the crucial function for the regulation of Cys biosynthesis in vivo. An Arabidopsis thaliana SAT was overexpressed in the cytosol of transgenic tobacco (Nicotiana tabacum) plants in either enzymatically active or inactive forms that were both shown to interact efficiently with endogenous tobacco OAS-TL proteins. Active SAT expression resulted in a 40-fold increase in SAT activity and strong increases in the reaction intermediate OAS as well as Cys, glutathione, Met, and total sulfur contents. However, inactive SAT expression produced much greater enhancing effects, including 30-fold increased Cys levels, attributable, apparently, to the competition of inactive transgenic SAT with endogenous tobacco SAT for binding to OAS-TL. Expression levels of tobacco SAT and OAS-TL remained unaffected. Flux control coefficients suggested that the accumulation of OAS and Cys in both types of transgenic plants was accomplished by different mechanisms. These data provide evidence that the CSC and its subcellular compartmentation play a crucial role in the control of Cys biosynthesis, a unique function for a plant metabolic protein complex.
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functional analysis of the Cysteine Synthase protein complex from plants structural biochemical and regulatory properties
Journal of Plant Physiology, 2006Co-Authors: Markus Wirtz, Rudiger HellAbstract:Cysteine synthesis in plants represents the final step of assimilatory sulfate reduction and the almost exclusive entry reaction of reduced sulfur into metabolism not only of plants, but also the human food chain in general. It is accomplished by the sequential reaction of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they form the hetero-oligomeric Cysteine Synthase complex (CSC). Recent evidence is reviewed that identifies the dual function of the CSC as a sensor and as part of a regulatory circuit that controls cellular sulfur homeostasis. Computational modeling of three-dimensional structures of plant SAT and OAS-TL based on the crystal structure of the corresponding bacterial enzymes supports quaternary conformations of SAT as a dimer of trimers and OAS-TL as a homodimer. These findings suggest an overall alpha6beta4 structure of the subunits of the plant CSC. Kinetic measurements of CSC dissociation triggered by the reaction intermediate O-acetylserine as well as CSC stabilization by sulfide indicate quantitative reactions that are suited to fine-tune the equilibrium between free and associated CSC subunits. In addition, in vitro data show that SAT requires binding to OAS-TL for full activity, while at the same time bound OAS-TL becomes inactivated. Since OAS concentrations inside cells increase upon sulfate deficiency, whereas sulfide concentrations most likely decrease, these data suggest the dissociation of the CSC in vivo, accompanied by inactivation of SAT and activation of OAS-TL function in their free homo-oligomer states. Biochemical evidence describes this protein-interaction based mechanism as reversible, thus closing the regulatory circuit. The properties of the CSC and its subunits are therefore consistent with models of positive regulation of sulfate uptake and reduction in plants by OAS as well as a demand-driven repression/de-repression by a sulfur intermediate, such as sulfide.
Kazuro Shiomi - One of the best experts on this subject based on the ideXlab platform.
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discovery of antiamebic compounds that inhibit Cysteine Synthase from the enteric parasitic protist entamoeba histolytica by screening of microbial secondary metabolites
Frontiers in Cellular and Infection Microbiology, 2018Co-Authors: Mihoko Mori, Tomoyoshi Nozaki, Ghulam Jeelani, Kenichi Nonaka, Atsuko Matsumoto, Satoshi Tsuge, Wataru Fukasawa, Kumiko Nakadatsukui, Satoshi ōmura, Kazuro ShiomiAbstract:Amebiasis is caused by infection with the parasite, Entamoeba histolytica. Although metronidazole has been used against amebiasis, it shows side effects and low efficacy against asymptomatic cyst carriers. Therefore, drugs with new mode of action or targets are urgently needed. L-Cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica. E. histolytica possesses the de novo L-Cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and Cysteine Synthase (CS). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established the new screening system to discover antiamebic compounds that target the de novo Cysteine biosynthesis. The screening allowed us to identify the compounds that differentially affect the growth of the trophozoites in the Cysteine-deprived media compared to the Cysteine-containing medium. A total of 431 compounds of the Kitasato Natural Products Library and 6,900 of microbial culture broth extracts were screened. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in Cysteine-deprived medium when compared to the growth in the Cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural unstability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed Cysteine deprivation-dependent antiamebic activity, although the IC50 value in the Cysteine-deprived medium was rather high (283 M). Pencolide also showed inhibitory activity against EhCSs with comparable IC50 values. These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is attacked by Michael donors including the thiol moiety of Cysteine. The Cysteine-adducts were detected by mass spectrometric analysis as predicted. Thus, we cannot exclude a possibility that the Cysteine-adducts of pencolide may contribute Cysteine-dependent toxicity of pencolide to the parasite. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a Cysteine-dependent manner with relatively low mammalian cytotoxicity.
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discovery of antiamebic compounds that inhibit Cysteine Synthase from the enteric parasitic protist entamoeba histolytica by screening of microbial secondary metabolites
Frontiers in Cellular and Infection Microbiology, 2018Co-Authors: Mihoko Mori, Tomoyoshi Nozaki, Ghulam Jeelani, Kenichi Nonaka, Atsuko Matsumoto, Satoshi Tsuge, Wataru Fukasawa, Kumiko Nakadatsukui, Satoshi ōmura, Kazuro ShiomiAbstract:Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-Cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-Cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and Cysteine Synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo Cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the Cysteine-deprived medium compared to the Cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the Cysteine-deprived medium when compared to the growth in the Cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed Cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of Cysteine than that in the presence of Cysteine), although the IC50 value in the Cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of Cysteine. The Cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the Cysteine-containing medium, the Cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the Cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of Cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a Cysteine-dependent manner with relatively low mammalian cytotoxicity.
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Data_Sheet_1_Discovery of Antiamebic Compounds That Inhibit Cysteine Synthase From the Enteric Parasitic Protist Entamoeba histolytica by Screening of Microbial Secondary Metabolites.pdf
2018Co-Authors: Mihoko Mori, Tomoyoshi Nozaki, Ghulam Jeelani, Kenichi Nonaka, Atsuko Matsumoto, Satoshi Tsuge, Wataru Fukasawa, Kumiko Nakada-tsukui, Satoshi Ōmura, Kazuro ShiomiAbstract:Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-Cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-Cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and Cysteine Synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo Cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the Cysteine-deprived medium compared to the Cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the Cysteine-deprived medium when compared to the growth in the Cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed Cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of Cysteine than that in the presence of Cysteine), although the IC50 value in the Cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of Cysteine. The Cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the Cysteine-containing medium, the Cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the Cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of Cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a Cysteine-dependent manner with relatively low mammalian cytotoxicity.
Rudiger Hell - One of the best experts on this subject based on the ideXlab platform.
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the redox sensitive module of cyclophilin 20 3 2 Cysteine peroxiredoxin and Cysteine Synthase integrates sulfur metabolism and oxylipin signaling in the high light acclimation response
Plant Journal, 2017Co-Authors: Sara Mareike Muller, Markus Wirtz, Andrea Viehhauser, Shanshan Wang, Wilena Telman, Michael Liebthal, Helena Schnitzer, Carsten Sticht, Carolina Delatorre, Rudiger HellAbstract:Summary The integration of redox- and reactive oxygen species-dependent signaling and metabolic activities are fundamental to plant acclimation to biotic and abiotic stresses. Previous data suggest the existence of a dynamically interacting module in the chloroplast stroma consisting of cyclophilin 20-3 (Cyp20-3), O-acetylserine(thiol)lyase B (OASTL-B), 2-Cysteine peroxiredoxins A/B (2-CysPrx) and serine acetyltransferase 2;1 (SERAT2;1). The functionality of this COPS module is influenced by redox stimuli and oxophytodienoic acid (OPDA) which is the precursor for jasmonic acid. The concept of an integrating function of these proteins in stress signaling was challenged by combining transcriptome and biochemical analyses in Arabidopsis mutants devoid of oastlB, serat 2;1, cyp20-3 and 2-cysprxA/B, and wildtype. Leaf transcriptomes were analyzed 6h after transfer to light intensity 10-fold in excess of growth light or under growth light. The survey of KEGG-based gene ontology groups showed common upregulation of translation- and protein homeostasis-associated transcripts under control conditions in all mutants compared to wildtype. The results revealed that the interference of the module was accompanied with disturbance of carbohydrate, sulfur and nitrogen metabolism and also citric acid cycle intermediates. Apart from common regulation, specific responses at the transcriptome and metabolite level linked Cyp20-3 to cell wall-bound carbohydrates and oxylipin signaling, and 2-CysPrx to photosynthesis, sugar and amino acid metabolism. Deletion of either OASTL-B or SERAT2;1 frequently induced antagonistic changes in biochemical or molecular features. Enhanced sensitivity of mutant seedlings to OPDA and leaf discs to NaHS-administration confirmed the presumed functional interference of the COPS module in redox and oxylipin signaling. This article is protected by copyright. All rights reserved.
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the redox sensitive module of cyclophilin 20 3 2 Cysteine peroxiredoxin and Cysteine Synthase integrates sulfur metabolism and oxylipin signaling in the high light acclimation response
Plant Journal, 2017Co-Authors: Sara Mareike Muller, Markus Wirtz, Andrea Viehhauser, Shanshan Wang, Wilena Telman, Michael Liebthal, Helena Schnitzer, Carsten Sticht, Carolina Delatorre, Rudiger HellAbstract:The integration of redox- and reactive oxygen species-dependent signaling and metabolic activities is fundamental to plant acclimation to biotic and abiotic stresses. Previous data suggest the existence of a dynamically interacting module in the chloroplast stroma consisting of cyclophilin 20-3 (Cyp20-3), O-acetylserine(thiol)lyase B (OASTL-B), 2-Cysteine peroxiredoxins A/B (2-CysPrx) and serine acetyltransferase 2;1 (SERAT2;1). The functionality of this COPS module is influenced by redox stimuli and oxophytodienoic acid (OPDA), which is the precursor for jasmonic acid. The concept of an integrating function of these proteins in stress signaling was challenged by combining transcriptome and biochemical analyses in Arabidopsis mutants devoid of oastlB, serat2;1, cyp20-3 and 2-cysprxA/B, and wild-type (WT). Leaf transcriptomes were analyzed 6 h after transfer to light intensity 10-fold in excess of growth light or under growth light. The survey of KEGG-based gene ontology groups showed common upregulation of translation- and protein homeostasis-associated transcripts under control conditions in all mutants compared with WT. The results revealed that the interference of the module was accompanied with disturbance of carbohydrate, sulfur and nitrogen metabolism, and also citric acid cycle intermediates. Apart from common regulation, specific responses at the transcriptome and metabolite level linked Cyp20-3 to cell wall-bound carbohydrates and oxylipin signaling, and 2-CysPrx to photosynthesis, sugar and amino acid metabolism. Deletion of either OASTL-B or SERAT2;1 frequently induced antagonistic changes in biochemical or molecular features. Enhanced sensitivity of mutant seedlings to OPDA and leaf discs to NaHS-administration confirmed the presumed functional interference of the COPS module in redox and oxylipin signaling.
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molecular biology biochemistry and cellular physiology of Cysteine metabolism in arabidopsis thaliana
The Arabidopsis Book, 2011Co-Authors: Rudiger Hell, Markus WirtzAbstract:Cysteine is one of the most versatile molecules in biology, taking over such different functions as catalysis, structure, regulation and electron transport during evolution. Research on Arabidopsis has contributed decisively to the understanding of Cysteine synthesis and its role in the assimilatory pathways of S, N and C in plants. The multimeric Cysteine Synthase complex is present in the cytosol, plastids and mitochondria and forms the centre of a unique metabolic sensing and signaling system. Its association is reversible, rendering the first enzyme of Cysteine synthesis active and the second one inactive, and vice-versa. Complex formation is triggered by the reaction intermediates of Cysteine synthesis in response to supply and demand and gives rise to regulation of genes of sulfur metabolism to adjust cellular sulfur homeostasis. Combinations of biochemistry, forward and reverse genetics, structural- and cell-biology approaches using Arabidopsis have revealed new enzyme functions and the unique pattern of spatial distribution of Cysteine metabolism in plant cells. These findings place the synthesis of Cysteine in the centre of the network of primary metabolism.
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dominant negative modification reveals the regulatory function of the multimeric Cysteine Synthase protein complex in transgenic tobacco
The Plant Cell, 2007Co-Authors: Markus Wirtz, Rudiger HellAbstract:Cys synthesis in plants constitutes the entry of reduced sulfur from assimilatory sulfate reduction into metabolism. The catalyzing enzymes serine acetyltransferase (SAT) and O-acetylserine (OAS) thiol lyase (OAS-TL) reversibly form the heterooligomeric Cys Synthase complex (CSC). Dominant-negative mutation of the CSC showed the crucial function for the regulation of Cys biosynthesis in vivo. An Arabidopsis thaliana SAT was overexpressed in the cytosol of transgenic tobacco (Nicotiana tabacum) plants in either enzymatically active or inactive forms that were both shown to interact efficiently with endogenous tobacco OAS-TL proteins. Active SAT expression resulted in a 40-fold increase in SAT activity and strong increases in the reaction intermediate OAS as well as Cys, glutathione, Met, and total sulfur contents. However, inactive SAT expression produced much greater enhancing effects, including 30-fold increased Cys levels, attributable, apparently, to the competition of inactive transgenic SAT with endogenous tobacco SAT for binding to OAS-TL. Expression levels of tobacco SAT and OAS-TL remained unaffected. Flux control coefficients suggested that the accumulation of OAS and Cys in both types of transgenic plants was accomplished by different mechanisms. These data provide evidence that the CSC and its subcellular compartmentation play a crucial role in the control of Cys biosynthesis, a unique function for a plant metabolic protein complex.
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functional analysis of the Cysteine Synthase protein complex from plants structural biochemical and regulatory properties
Journal of Plant Physiology, 2006Co-Authors: Markus Wirtz, Rudiger HellAbstract:Cysteine synthesis in plants represents the final step of assimilatory sulfate reduction and the almost exclusive entry reaction of reduced sulfur into metabolism not only of plants, but also the human food chain in general. It is accomplished by the sequential reaction of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they form the hetero-oligomeric Cysteine Synthase complex (CSC). Recent evidence is reviewed that identifies the dual function of the CSC as a sensor and as part of a regulatory circuit that controls cellular sulfur homeostasis. Computational modeling of three-dimensional structures of plant SAT and OAS-TL based on the crystal structure of the corresponding bacterial enzymes supports quaternary conformations of SAT as a dimer of trimers and OAS-TL as a homodimer. These findings suggest an overall alpha6beta4 structure of the subunits of the plant CSC. Kinetic measurements of CSC dissociation triggered by the reaction intermediate O-acetylserine as well as CSC stabilization by sulfide indicate quantitative reactions that are suited to fine-tune the equilibrium between free and associated CSC subunits. In addition, in vitro data show that SAT requires binding to OAS-TL for full activity, while at the same time bound OAS-TL becomes inactivated. Since OAS concentrations inside cells increase upon sulfate deficiency, whereas sulfide concentrations most likely decrease, these data suggest the dissociation of the CSC in vivo, accompanied by inactivation of SAT and activation of OAS-TL function in their free homo-oligomer states. Biochemical evidence describes this protein-interaction based mechanism as reversible, thus closing the regulatory circuit. The properties of the CSC and its subunits are therefore consistent with models of positive regulation of sulfate uptake and reduction in plants by OAS as well as a demand-driven repression/de-repression by a sulfur intermediate, such as sulfide.
Gunter Schneider - One of the best experts on this subject based on the ideXlab platform.
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pyridoxal phosphate dependent mycobacterial Cysteine Synthases structure mechanism and potential as drug targets
Biochimica et Biophysica Acta, 2015Co-Authors: Robert Schnell, Dharmarajan Sriram, Gunter SchneiderAbstract:The alarming increase of drug resistance in Mycobacterium tuberculosis strains poses a severe threat to human health. Chemotherapy is particularly challenging because M. tuberculosis can persist in the lungs of infected individuals; estimates of the WHO indicate that about 1/3 of the world population is infected with latent tuberculosis providing a large reservoir for relapse and subsequent spread of the disease. Persistent M. tuberculosis shows considerable tolerance towards conventional antibiotics making treatment particularly difficult. In this phase the bacilli are exposed to oxygen and nitrogen radicals generated as part of the host response and redox-defense mechanisms are thus vital for the survival of the pathogen. Sulfur metabolism and de novo Cysteine biosynthesis have been shown to be important for the redox homeostasis in persistent M. tuberculosis and these pathways could provide promising targets for novel antibiotics for the treatment of the latent form of the disease. Recent research has provided evidence for three de novo metabolic routes of Cysteine biosynthesis in M. tuberculosis, each with a specific PLP dependent Cysteine Synthase with distinct substrate specificities. In this review we summarize our present understanding of these pathways, with a focus on the advances on functional and mechanistic characterization of mycobacterial PLP dependent Cysteine Synthases, their role in the various pathways to Cysteine, and first attempts to develop specific inhibitors of mycobacterial Cysteine biosynthesis. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
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the c terminal of cysm from mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity
FEBS Letters, 2009Co-Authors: Daniel Agren, Robert Schnell, Gunter SchneiderAbstract:A new crystal structure of the dimeric Cysteine Synthase CysM from Mycobacterium tuberculosis reveals an open and a closed conformation of the enzyme. In the closed conformation the five carboxy-terminal amino acid residues are inserted into the active site cleft. Removal of this segment results in a decreased lifetime of the α-aminoacrylate reaction intermediate, an increased sensitivity to oxidants such as hydrogen peroxide, and loss of substrate selectivity with respect to the sulfur carrier thiocarboxylated CysO. These results highlight features of CysM that might be of particular importance for Cysteine biosynthesis under oxidative stress in M. tuberculosis.
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Cysteine Synthase cysm of mycobacterium tuberculosis is an o phosphoserine sulfhydrylase evidence for an alternative Cysteine biosynthesis pathway in mycobacteria
Journal of Biological Chemistry, 2008Co-Authors: Daniel Agren, R Schnell, Wulf Oehlmann, Mahavir Singh, Gunter SchneiderAbstract:Abstract The biosynthesis of Cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the Cysteine Synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific Cysteine Synthase. CysM belongs to the fold type II pyridoxal 5′-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-A resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity. Replacement of this residue results in a significant loss of specificity for O-phosphoserine. Notably, reactions with sulfur donors are not affected by the amino acid replacement. The specificity of CysM toward O-phosphoserine together with the previously established novel mode of sulfur delivery via thiocarboxylated CysO (Burns, K. E., Baumgart, S., Dorrestein, P. C., Zhai, H., McLafferty, F. W., and Begley, T. P. (2005) J. Am. Chem. Soc. 127, 11602–11603) provide strong evidence for an O-phosphoserine-based Cysteine biosynthesis pathway in M. tuberculosis that is independent of both O-acetylserine and the sulfate reduction pathway. The existence of an alternative biosynthetic pathway to Cysteine in this pathogen has implications for the design strategy aimed at inhibition of this metabolic route.
