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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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Edited by:
2015Co-Authors: Sílvia Tavares, Markus Wirtz, Rudiger Hell, Marcel P. Beier, Jochen Bogs, Sara Amâncio, Hanjo A. Hellmann, Mutsumi Watanabe, Max PlanckAbstract:doi: 10.3389/fpls.2015.00074 Characterization of the Serine Acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plant
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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.
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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drought stress in maize causes differential acclimation responses of glutathione and sulfur metabolism in leaves and roots
BMC Plant Biology, 2016Co-Authors: Markus Wirtz, Nisar Ahmad, Mario Malagoli, Ruediger HellAbstract:Drought is the most important environmental stress that limits crop yield in a global warming world. Despite the compelling evidence of an important role of oxidized and reduced sulfur-containing compounds during the response of plants to drought stress (e.g. sulfate for stomata closure or glutathione for scavenging of reactive oxygen species), the assimilatory sulfate reduction pathway is almost not investigated at the molecular or at the whole plant level during drought. In the present study, we elucidated the role of assimilatory sulfate reduction in roots and leaves of the staple crop maize after application of drought stress. The time-resolved dynamics of the adaption processes to the stress was analyzed in a physiological relevant situation –when prolonged drought caused significant oxidation stress but root growth should be maintained. The allocation of sulfate was significantly shifted to the roots upon drought and allowed for significant increase of thiols derived from sulfate assimilation in roots. This enabled roots to produce biomass, while leaf growth was stopped. Accumulation of harmful reactive oxygen species caused oxidation of the glutathione pool and decreased glutathione levels in leaves. Surprisingly, flux analysis using [35S]-sulfate demonstrated a significant down-regulation of sulfate assimilation and cysteine synthesis in leaves due to the substantial decrease of Serine Acetyltransferase activity. The insufficient cysteine supply caused depletion of glutathione pool in spite of significant transcriptional induction of glutathione synthesis limiting GSH1. Furthermore, drought impinges on transcription of membrane-localized sulfate transport systems in leaves and roots, which provides a potential molecular mechanism for the reallocation of sulfur upon prolonged water withdrawal. The study demonstrated a significant and organ-specific impact of drought upon sulfate assimilation. The sulfur metabolism related alterations at the transcriptional, metabolic and enzyme activity level are consistent with a promotion of root growth to search for water at the expense of leaf growth. The results provide evidence for the importance of antagonistic regulation of sulfur metabolism in leaves and roots to enable successful drought stress response at the whole plant level.
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Metabolites quantification and supplementation.
2016Co-Authors: Xin-yuan Huang, Markus Wirtz, Anna Koprivova, Dai-yin Chao, John Danku, Steffen Müller, Francisco J. Sandoval, Hermann Bauwe, Sanja Roje, Brian DilkesAbstract:(A) Schematic representation of sulphur assimilation in A. thaliana. Colour squares above the metabolites represent the log2 value of the msa1-1/WT Col-0 ratio of the concentration of each metabolite. APR: APS reductase; APS: adenosine 5’-phosphosulfate; ATPS: ATP sulfurylase; CBL: cystathionine β-lyase; CGS: cystathionine γ-synthase; Cyst: cystathionine; γ-ECS: γ-glutamylcysteine synthetase; γ-GluCys: γ-glutamylcysteine; GSHS: glutathione synthetase; Hcy: homocysteine; MS: methionine synthase; OAS: O-acetylSerine; OAS-TL: OAS(thiol)lyase; SAT: Serine Acetyltransferase; SAMS, S-adenosylmethionine synthetase; SiR: sulphite reductase; SHM: Serine hydroxymethyltransferase. (B-G) Measurement of sulphur-related metabolites. Plants were grown on agar solidified MGRL media under S sufficient (S1500) or S deficient (S0) conditions. Metabolites were extracted from shoots and roots and quantified by HPLC. Data are presented as means ± SD (n = 3). *, P ≤ 0.05; **, P ≤ 0.01, Student’s t test. (H-I) The concentrations of SAM and MTA in the shoots and roots of WT Col-0 and msa1-1 grown under S sufficient condition. (J) Total S in the shoots of WT Col-0 and msa1-1 grown under S sufficient condition without (CK) or with SAM added to the growth medium. Data in (B-J) are presented as means ± SD (n = 3 in (B-G), n = 5 in (H-I), and n = 6 in (J)). * and ** in (B-J) indicate values significantly different between WT Col-0 and msa1-1 mutant at P ≤ 0.05 and P ≤ 0.01, respectively (Student’s t test). DW, dry weight. CK, control.
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Edited by:
2015Co-Authors: Sílvia Tavares, Markus Wirtz, Rudiger Hell, Marcel P. Beier, Jochen Bogs, Sara Amâncio, Hanjo A. Hellmann, Mutsumi Watanabe, Max PlanckAbstract:doi: 10.3389/fpls.2015.00074 Characterization of the Serine Acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plant
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 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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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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Characterization of Transsulfuration and Cysteine Biosynthetic Pathways in the Protozoan Hemoflagellate, Trypanosoma cruzi: ISOLATION AND MOLECULAR CHARACTERIZATION OF CYSTATHIONINE β-SYNTHASE AND Serine Acetyltransferase FROMTRYPANOSOMA
The Journal of biological chemistry, 2000Co-Authors: Tomoyoshi Nozaki, Mihoko Imada, Yasuo Shigeta, Yumiko Saito-nakano, Warren D. KrugerAbstract:Abstract Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis. We cloned and characterized cDNA encoding cystathionine β-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine.T. cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes. The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has Serine sulfhydrylase and cysteine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiaeand Escherichia coli demonstrates that the CBS and CS activities are functional in vivo. Enzymatic studies onT. cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression. We also cloned and characterized cDNA encoding Serine Acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a recombinant T. cruzi SAT showed allosteric inhibition by l-cysteine,l-cystine, and, to a lesser extent, glutathione. Together, these studies demonstrate the T. cruzi is a unique protist in possessing both transsulfuration and sulfur assimilatory pathways.
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cloning and biochemical characterization of genes encoding two isozymes of cysteine synthase from entamoeba dispar
Molecular and Biochemical Parasitology, 2000Co-Authors: Tomoyoshi Nozaki, Masaharu Tokoro, Mihoko Imada, Yumiko Saito, Yoko Abe, Yasuo Shigeta, Tsutomu TakeuchiAbstract:Entamoeba histolytica and E. dispar are two morphologically indistinguishable protist parasites that inhabit the human intestine [1]. These two species are closely related and share a variety of biological and biochemical characteristics [2]. However, it has been well established that they differ in clinical importance: E. dispar trophozoites do not invade tissues or at most produce superficial erosion of the colonic mucosa in the mammalian host, whereas E. histolytica occasionally invades and destroys intestinal and extraintestinal tissues and causes diseases such as amoebic dysentery and liver abscess. Many genes encoding putative pathogenic factors from E. histolytica have been cloned and characterized (see e.g. [3] for review), suggesting that virulence is likely to be multifactorial. However, except for one isozyme of cysteine proteinases (CP5) [4], these putative pathogenic factors are present in both E. histolytica and E. dispar [5–8]. Thus, factors that differentiate the pathogenic E. histolytica from the nonpathogenic E. dispar have not been identified except for CP5, which has been shown to be involved in liver abscess formation by E. histolytica trophozoites [9]. We previously showed that Entamoebae are capable of de novo synthesis of L-cysteine, unlike their mammalian hosts [10–12]. This pathway plays an important role in sulfur assimilation. Amoeba trophozoites transport sulfate intracellularly, activate and reduce it, and finally fix it into Abbre6iations: SAT, Serine Acetyltransferase; CS, cysteine synthase; ORF, open reading frame; PCR, polymerase chain reaction. Note : The nucleotide sequences data reported in this paper are available in the DDBJ, EMBL, and GenBank databases under the accession numbers AB028631 and AB028632. * Corresponding author. Tel.: +81-3-52851111 ext. 2733; fax: +81-3-52851173. E-mail address: nozaki@nih.go.jp. (T. Nozaki)
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characterization of the gene encoding Serine Acetyltransferase a regulated enzyme of cysteine biosynthesis from the protist parasites entamoeba histolytica and entamoeba dispar regulation and possible function of the cysteine biosynthetic pathway in
Journal of Biological Chemistry, 1999Co-Authors: Tomoyoshi Nozaki, Takashi Asai, Seiki Kobayashi, Lidya B Sanchez, Miki Nakazawa, Tsutomu TakeuchiAbstract:Abstract The enteric protist parasitesEntamoeba histolytica and Entamoeba disparpossess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production ofl-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme Serine Acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36–52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT fromSynechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by l-cysteine in a competitive manner withl-Serine; 2) inhibition by l-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.
Kazuki Saito - One of the best experts on this subject based on the ideXlab platform.
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metabolic engineering of sulfur assimilation in plants
2007Co-Authors: Masaaki Noji, Kazuki SaitoAbstract:Sulfur and sulfur-containing products play important roles in plant cells. In order to enhance the ability of the production of sulfur-containing compounds by the metabolic engineering of sulfur assimilation in plants, we constructed transgenic plants over-expressing cysteine synthase (CSase), or Serine Acetyltransferase (SATase) gene, key enzymes for cysteine biosynthesis in plants. Transgenic plants over-expressing CSase gene were highly tolerant to sulfur dioxide, sulfite, oxidative stress, and cadmium. Besides, transgenic plants over-expressing SATase gene showed the over-accumulation of sulfur-containing compounds, cysteine and glutathione (GSH). These results indicate that the over-expression of CSase or SATase gene is promising for the metabolic engineering of sulfur assimilation in plants
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Molecular and biochemical analysis of Serine Acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants.
Amino acids, 2002Co-Authors: Masaaki Noji, Kazuki SaitoAbstract:Serine Acetyltransferase (SATase) and cysteine synthase (O-acetylSerine (thiol)-lyase) (CSase) are committed in the final step of cysteine biosynthesis. Six cDNA clones encoding SATase have been isolated from several plants, e.g. watermelon, spinach, Chinese chive and Arabidopsis thaliana. Feedback-inhibition pattern and subcellular localization of plant SATases were evaluated. Two types of SATase that differ in their sensitivity to the feedback inhibition by l-cysteine were found in plants. In Arabidopsis, cytosolic SATase was inhibited by l-cysteine at a physiological concentration in an allosteric manner, but the plastidic and mitochondrial forms were not subjected to this feedback regulation. These results suggest that the regulation of cysteine biosynthesis through feedback inhibition may differ depending on the subcellular compartment. The allosteric domain responsible for l-cysteine inhibition was characterized, using several SATase mutants. The single change of amino acid residue, glycine-277 to cysteine, in the C-terminal region of watermelon SATase caused a significant decrease of the feedback-inhibition sensitivity of watermelon SATase. We made the transgenic Arabidopsis overexpressing point-mutated watermelon SATase gene whose product was not inhibited by l-cysteine. The contents of OAS, cysteine, and glutathione in transgenic Arabidopsis were significantly increased as compared to the wild-type Arabidopsis. Transgenic tobacco (Nicotiana tabacum) (F1) plants with enhanced CSase activities both in the cytosol and in the chloroplasts were generated by cross-fertilization of two transgenic tobacco expressing either cytosolic CSase or chloroplastic CSase. Upon fumigation with 0.1 μL L−1 sulfur dioxide, both the cysteine and glutathione contents in leaves of F1 plants were increased significantly, but not in leaves of non-transformed control plants. These results indicated that both SATase and CSase play important roles in cysteine biosynthesis and its regulation in plants.
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molecular cloning and functional characterization of cdnas encoding cysteine synthase and Serine Acetyltransferase that may be responsible for high cellular cysteine content in allium tuberosum
Gene, 2000Co-Authors: Yasuomi Urano, Masaaki Noji, Tomofumi Manabe, Kazuki SaitoAbstract:Abstract The plants belonging to the genus Allium are known to accumulate sulfur-containing secondary compounds that are derived from cysteine. Here, we report on molecular cloning and functional characterization of two cDNAs that encode Serine Acetyltransferase and cysteine synthase from A. tuberosum (Chinese chive). The cDNA for Serine Acetyltransferase encodes an open reading frame of 289 amino acids, of which expression could complement the lacking of cysE gene for endogenous Serine Acetyltransferase in Escherichia coli. The cDNA for cysteine synthase encodes an open reading frame of 325 amino acids, of which expression in the E. coli lacking endogenous cysteine synthase genes could functionally rescue the growth without addition of cysteine. Both deduced proteins seem to be localized in cytosol, judging from their primary structures. Northern blot analysis indicated that both transcripts accumulated in almost equal levels in leaves and root of green and etiolated seedlings of A. tuberosum. The activity of recombinant Serine Acetyltransferase produced from the cDNA was inhibited by l -cysteine, which is the end-product of the pathway; however, the sensitivity to cysteine (48.7 μM of the concentration for 50% inhibition, ic 50) was fairly low compared with that of previously reported Serine Acetyltransferases (∼5 μM ic 50) from various plants. In A. tuberosum, the cellular content of cysteine was several-fold higher than those in Arabidopsis thaliana and tobacco. This higher concentration of cysteine in A. tuberosum is likely due to the lower sensitivity of feedback inhibition of Serine Acetyltransferase to cysteine.
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regulation of sulfate transport and synthesis of sulfur containing amino acids
Current Opinion in Plant Biology, 2000Co-Authors: Kazuki SaitoAbstract:Recent research indicates that several sulfate transporters - exhibiting different tissue specificities and modes of expression - may play distinct roles in sulfate uptake within specific tissues and in long-distance sulfate translocation. The transcription levels of particular genes and feedback inhibition of Serine Acetyltransferase play major roles in regulating sulfur assimilation and cysteine synthesis. O-acetylSerine and glutathione presumably act within the cysteine synthesis pathway as derepressor and repressor, respectively. A unique autoregulatory mechanism that stabilizes mRNA levels has recently been proposed for the regulation of methionine synthesis.
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regulation of sulfur assimilation in higher plants a sulfate transporter induced in sulfate starved roots plays a central role in arabidopsis thaliana
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Hideki Takahashi, Mami Yamazaki, Noriko Sasakura, Akiko Watanabe, Thomas Leustek, Janice De Almeida Engler, Gilbert Engler, Marc Van Montagu, Kazuki SaitoAbstract:Proton/sulfate cotransporters in the plasma membranes are responsible for uptake of the environmental sulfate used in the sulfate assimilation pathway in plants. Here we report the cloning and characterization of an Arabidopsis thaliana gene, AST68, a new member of the sulfate transporter gene family in higher plants. Sequence analysis of cDNA and genomic clones of AST68 revealed that the AST68 gene is composed of 10 exons encoding a 677-aa polypeptide (74.1 kDa) that is able to functionally complement a Saccharomyces cerevisiae mutant lacking a sulfate transporter gene. Southern hybridization and restriction fragment length polymorphism mapping confirmed that AST68 is a single-copy gene that maps to the top arm of chromosome 5. Northern hybridization analysis of sulfate-starved plants indicated that the steady-state mRNA abundance of AST68 increased specifically in roots up to 9-fold by sulfate starvation. In situ hybridization experiments revealed that AST68 transcripts were accumulated in the central cylinder of sulfate-starved roots, but not in the xylem, endodermis, cortex, and epidermis. Among all the structural genes for sulfate assimilation, sulfate transporter (AST68), APS reductase (APR1), and Serine Acetyltransferase (SAT1) were inducible by sulfate starvation in A. thaliana. The sulfate transporter (AST68) exhibited the most intensive and specific response in roots, indicating that AST68 plays a central role in the regulation of sulfate assimilation in plants.
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Modulation of Escherichia coli Serine Acetyltransferase catalytic activity in the cysteine synthase complex.
FEBS letters, 2017Co-Authors: Roberto Benoni, Stefano Bettati, Andrea Mozzarelli, Christopher S Hayes, Omar De Bei, Gianluca Paredi, Nina Franko, Barbara CampaniniAbstract:In bacteria and plants, Serine Acetyltransferase (CysE) and O-acetylSerine sulfhydrylase-A sulfhydrylase (CysK) collaborate to synthesize l-Cys from l-Ser. CysE and CysK bind one another with high affinity to form the cysteine synthase complex (CSC). We demonstrate that bacterial CysE is activated when bound to CysK. CysE activation results from the release of substrate inhibition, with the Ki for l-Ser increasing from 4 mm for free CysE to 16 mm for the CSC. Feedback inhibition of CysE by l-Cys is also relieved in the bacterial CSC. These findings suggest that the CysE active site is allosterically altered by CysK to alleviate substrate and feedback inhibition in the context of the CSC.
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moonlighting o acetylSerine sulfhydrylase new functions for an old protein
Biochimica et Biophysica Acta, 2015Co-Authors: Barbara Campanini, Stefano Bettati, Roberto Benoni, Christina M Beck, Christopher S Hayes, Andrea MozzarelliAbstract:O-acetylSerine sulfhydrylase A (CysK) is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with Serine Acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This "cysteine synthase" complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional "moonlighting" activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode Caenorhabditis elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
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Fine tuning of the active site modulates in the interaction of O-acetylSerine sulfhydryase isozymes with Serine acetyl transferase
'Elsevier BV', 2013Co-Authors: Spyrakis Francesca, Enea Salsi, Mozzarelli Andrea, Felici Paolo, S. A. Bayden, R. Miggiano, E. G. Kellogg, Cozzini Pietro, F. P. Cook, Barbara CampaniniAbstract:O-acetylSerine sulfhydrylase (OASS) catalyzes the synthesis of l-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with Serine Acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein-peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3-P4-P5 for the strength of binding, and P1-P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with K(diss) in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents
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the multifaceted pyridoxal 5 phosphate dependent o acetylSerine sulfhydrylase
Biochimica et Biophysica Acta, 2011Co-Authors: Andrea Mozzarelli, Francesca Spyrakis, Ratna Singh, Barbara Campanini, Enea Salsi, Samanta Raboni, Stefano Bettati, Vidya Prasanna Kumar, Paul F. CookAbstract:Abstract Cysteine is the final product of the reductive sulfate assimilation pathway in bacteria and plants and serves as the precursor for all sulfur-containing biological compounds, such as methionine, S-adenosyl methionine, iron–sulfur clusters and glutathione. Moreover, in several microorganisms cysteine plays a role as a reducing agent, eventually counteracting host oxidative defense strategies. Cysteine is synthesized by the PLP-dependent O-acetylSerine sulfhydrylase, a dimeric enzyme belonging to the fold type II, catalyzing a beta-replacement reaction. In this review, the spectroscopic properties, catalytic mechanism, three-dimensional structure, conformational changes accompanying catalysis, determinants of enzyme stability, role of selected amino acids in catalysis, and the regulation of enzyme activity by ligands and interaction with Serine Acetyltransferase, the preceding enzyme in the biosynthetic pathway, are described. Given the key biological role played by O-acetylSerine sulfhydrylase in bacteria, inhibitors with potential antibiotic activity have been developed. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.
