Dan Sato

Tokyo Metropolitan University, Tokyo, Tokyo-to, Japan

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Publications (28)86.73 Total impact

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    ABSTRACT: Whole genome or transcriptome information provides the annotation of genes and proteins and predicts metabolic pathways, but unequivocal demonstration of the functionalities of the enzymes and metabolic pathways remains challenging. Because nearly 56 % of the Entamoeba histolytica genes remain unannotated, correlative “omics” analyses of genomics, transcriptomics, proteomics, and biochemical metabolic profiling can be useful in uncovering new, or poorly understood, metabolisms and metabolic pathways. Current understanding of metabolic pathways constructed by genes and pathway predictions are based on homology search of the genome, transcriptome, and proteome databases and conventional biochemical demonstration of enzymatic activities. However, it is well known that there are large disparities between the pathways predicted in silico and the pathways actually operating in vivo. Thus, it is important to demonstrate the presence and kinetics (flow or flux) of the metabolites involved in the pathways. To this end, a variety of analytical methods and platforms for metabolomics and metabolite profiling has been developed, in which intracellular and extracellular metabolites can be selectively or globally analyzed. Global metabolomics analysis of Entamoeba histolytica under environmental stress conditions, in different life-cycle stages, and heterogenic (i.e., clinical) isolates, should potentially uncover unpredictable metabolic pathways, interaction and regulation of pathways, and also directly demonstrate the role of individual genes on metabolic pathways, and thus helps our understanding of the physiological and biological roles of metabolic pathways and a network of regulatory interactions between them. Metabolomics of Entamoeba is still in its infancy and only a handful of studies have been reported thus far. In this chapter, we summarize a few examples of the application of metabolomics, combined with transcriptomic analysis, to the analysis of global changes in metabolism in response to three representative physiological conditions: encystation, oxidative stress, and cysteine deprivation. We also discuss future applications of metabolomics to understand the biology and pathogenesis of E. histolytica. Furthermore, because major metabolic differences between the parasite and its host provide rational drug targets, which are either selectively present in pathogens or highly divergent from humans, multi-“omics” approaches, including metabolomics, should lead to important discoveries of unique exploitable metabolic networks crucial to develop new effective drugs against amebiasis.
    Amebiasis: Biology and Pathogenesis of Entamoeba, IV edited by Tomoyoshi Nozaki, Alok Bhattacharya, 11/2014: chapter Metabolomic Analysis of Entamoeba Biology: pages 331-349; Springer Japan., ISBN: 978-4-431-55199-7
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    ABSTRACT: Aphid infestations can cause severe decreases in soybean (Glycine max [L.] Merr.) yield. Since planting aphid-resistant soybean strains is a promising approach for pest control, understanding the resistance mechanisms employed by aphids is of considerable importance. We compared aphid resistance in seven soybean strains and found that strain Tohoku149 was the most resistant to the foxglove aphid, Aulacorthum solani Kaltenbach. We subsequently analyzed the metabolite profiles of aphids cultured on the leaves of resistant and susceptible soybean strains using capillary electrophoresis-time-of-flight mass spectrometry. Our findings showed that the metabolite profiles of several amino acids, glucose 6-phosphate, and components of the tricarboxylic acid cycle were similar in aphids reared on Tohoku149 leaves and in aphids maintained under conditions of starvation, suggesting that Tohoku149 is more resistant to aphid feeding. Compared to susceptible strains, we also found that two methylated metabolites, S-methylmethionine and trigonelline, were either not detected or decreased in aphids reared on Tohoku149 plants. Since these metabolites function as important sulfur transporters in phloem sap and osmoprotectants involved in salt and drought stress, respectively, aphid-resistance is considered to be related to sulfur metabolism and methylation. These results contribute to an increase in our understanding of soybean aphid resistance mechanisms at the molecular level.
    Molecular BioSystems 02/2014; · 3.35 Impact Factor
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    ABSTRACT: l-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled l-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of l-cysteine in E. histolytica. [U-(13)C3, (15)N]l-cysteine was rapidly metabolized into three unknown metabolites, besides l-cystine and l-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of l-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of l-cysteine. Liberation of l-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these l-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress.
    mBio 01/2014; 5(6). · 6.88 Impact Factor
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    ABSTRACT: Aphid infection reduces soybean (Glycine max [L.] Merr.) yield. Consequently, cultivation of aphid-resistant strains is a promising approach to pest control, and understanding the resistance mechanism is of importance. Here, we characterized the resistance of soybeans to foxglove aphid, Aulacorthum solani Kaltenbach, at the metabolite level. First, we evaluated aphid mortality and settlement rates on the leaves of two soybean strains, 'Tohoku149' and 'Suzuyutaka', and found that the former had strong resistance soon after introduction of the aphids. The metabolomic response to aphid introduction was analyzed using capillary electrophoresis-time-of-flight mass spectrometry. We found the following three features in the profiles: (1) concentrations of citrate, amino acids, and their intermediates were intrinsically higher for Tohoku149 than Suzuyutaka, (2) concentrations of several metabolites producing secondary metabolites, such as flavonoids and alkaloids, drastically changed 6h after aphid introduction, and (3) concentrations of TCA cycle metabolites increased in Tohoku149 48h after aphid introduction. We also profiled free amino acids in aphids reared on both soybean strains and under starvation, and found that the profile of the aphids on Tohoku149 was similar to that of the starved aphids, but different to that of aphids on Suzuyutaka. These tests confirmed that aphids suck phloem sap even from Tohoku149. This study demonstrates the metabolomic profiles of both soybean strains and aphids, which will contribute to the molecular level understanding of mechanisms of soybean resistance to aphids.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 03/2013; 925C:95-103. · 2.78 Impact Factor
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    ABSTRACT: The insect cuticle is non-cellular matrix secreted from a monolayer of epidermal cells. After abrasion of the larval cuticle of the silkworm, Bombyx mori, a protein with molecular mass of 135 kDa is newly detected in the cuticle. Mass spectrometric analysis of the tryptic fragments from this protein revealed that the 135-kDa protein is encoded by the Cb10 gene. In the predicted amino acid sequence of Cb10, three repeated motifs with [YxGGFGGppG(L/V)L] sequence are found in the C-terminal region. In addition to the repeated motifs, Cb10 has seventeen CxxxxC motifs randomly distributed throughout the polypeptide chain and serine rich region at N-terminal region. The Cb10 gene is strongly expressed in epidermal cells after pupal ecdysis, and its expression in the larval epidermal cells is induced not only by cuticular abrasion, but also by bacterial infection. These expression patterns suggest some specific roles of this protein in pupal cuticle formation and defense reactions.
    Insect biochemistry and molecular biology 01/2013; · 3.25 Impact Factor
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    ABSTRACT: NAD(H) kinase catalyzes the phosphorylation of NAD(H) to form NADP(H) using ATP or inorganic polyphosphate as a phosphoryl donor. While the enzyme is conserved throughout prokaryotes and eukaryotes, remarkable differences in kinetic parameters including substrate preference, cation dependence, and physiological roles exist among the organisms. In the present study, we biochemically characterized NAD(H) kinase from the anaerobic/microaerophilic fermentative protozoan parasite Entamoeba histolytica, which lacks the conventional mitochondria capable of oxidative phosphorylation, leading to ATP. The kinetic properties of E. histolytica NAD(H) kinase recombinantly produced in Escherichia coli showed remarkable differences from those in bacteria and higher eukaryotes. Entamoeba NAD(H) kinase preferred NADH to NAD+ as the phosphoryl acceptor, utilized nucleoside triphosphates including ATP, GTP and deoxyATP, but not nucleoside di-, mono-phosphates, or inorganic polyphosphates, as the phosphoryl donor. To further understand the physiological roles in E. histolytica, we generated a stable transformant overexpressing NAD(H) kinase. Overexpression of NAD(H) kinase resulted in a 1.6-2 fold increase in the NADPH and NADP+ concentrations, a 40% reduction of the intracellular concentration of reactive oxygen species, and also led to increased tolerance toward hydrogen peroxide. These data, together with the essentially of NAD(H) kinase gene, underscore its significance as an NADP(H)-producing enzyme in this organism, and should help in designing of drugs targeting this enzyme.
    Biochimie 10/2012; · 3.14 Impact Factor
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    ABSTRACT: Entamoeba histolytica, a microaerophilic enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. Trophozoites of E. histolytica are exposed to a variety of reactive oxygen and nitrogen species during infection. Since E. histolytica lacks key components of canonical eukaryotic anti-oxidative defense systems, such as catalase and glutathione system, alternative not-yet-identified anti-oxidative defense strategies have been postulated to be operating in E. histolytica. In the present study, we investigated global metabolic responses in E. histolytica in response to H(2)O(2)- and paraquat-mediated oxidative stress by measuring charged metabolites on capillary electrophoresis and time-of-flight mass spectrometry. We found that oxidative stress caused drastic modulation of metabolites involved in glycolysis, chitin biosynthesis, and nucleotide and amino acid metabolism. Oxidative stress resulted in the inhibition of glycolysis as a result of inactivation of several key enzymes, leading to the redirection of metabolic flux towards glycerol production, chitin biosynthesis, and the non-oxidative branch of the pentose phosphate pathway. As a result of the repression of glycolysis as evidenced by the accumulation of glycolytic intermediates upstream of pyruvate, and reduced ethanol production, the levels of nucleoside triphosphates were decreased. We also showed for the first time the presence of functional glycerol biosynthetic pathway in E. histolytica as demonstrated by the increased production of glycerol 3-phosphate and glycerol upon oxidative stress. We proposed the significance of the glycerol biosynthetic pathway as a metabolic anti-oxidative defense system in E. histolytica.
    PLoS Neglected Tropical Diseases 09/2012; 6(9):e1831. · 4.57 Impact Factor
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    ABSTRACT: Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.
    Bioscience Biotechnology and Biochemistry 07/2012; 76(7):1275-84. · 1.27 Impact Factor
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    ABSTRACT: Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst). We also found remarkable induction of γ-aminobutyric acid (GABA) during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause.
    PLoS ONE 01/2012; 7(5):e37740. · 3.53 Impact Factor
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    ABSTRACT: Drug resistance in parasitic protozoa is an obstacle to successful chemotherapy. Understanding how pathogens respond to drugs is crucial in preventing resistance. Previously, we have shown that in Entamoeba histolytica, methionine γ-lyase (EhMGL) downregulation results in trifluoromethionine resistance. The transcriptional response, however, of this parasite to the drug is not known. In this study, we used microarray analysis to determine whether additional genes are involved. The expression profiles of 9230 genes in wild-type and trifluoromethionine-resistant strains were compared. Episomal overexpression of EhBspA1 was performed to verify its role in trifluoromethionine resistance. The transcriptomes of a trifluoromethionine-resistant strain cultured with or without trifluoromethionine, an EhMGL gene-silenced strain, a strain with reduced susceptibility to metronidazole and a wild-type strain under cysteine-deprived conditions were compared to determine the specificity of the changes observed in the trifluoromethionine-resistant strain. The expression of 35 genes differed at least 3-fold between trifluoromethionine-resistant and wild-type strains. Some of the genes play roles in metabolism, the stress response and gene regulation. EhMGL and EhBspA1 were found to be highly downregulated and upregulated, respectively. Overexpression of EhBspA1 conferred partial resistance to trifluoromethionine. Comparative transcriptome analysis showed that genes modulated in trifluoromethionine-resistant strains were specific. E. histolytica has few known resistance mechanisms against drugs. In this study, we showed that aside from EhMGL downregulation, induction of EhBspA1 plays a role in trifluoromethionine resistance. We also showed a unique set of induced genes that could represent the signature profile of trifluoromethionine resistance in E. histolytica.
    Journal of Antimicrobial Chemotherapy 11/2011; 67(2):375-86. · 5.34 Impact Factor
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    ABSTRACT: Mitochondrion-related organelles, mitosomes and hydrogenosomes, are found in a phylogenetically broad range of organisms. Their components and functions are highly diverse. We have previously shown that mitosomes of the anaerobic/microaerophilic intestinal protozoan parasite Entamoeba histolytica have uniquely evolved and compartmentalized a sulfate activation pathway. Although this confined metabolic pathway is the major function in E. histolytica mitosomes, their physiological role remains unknown. In this study, we examined the phenotypes of the parasites in which genes involved in the mitosome functions were suppressed by gene silencing, and showed that sulfate activation in mitosomes is important for sulfolipid synthesis and cell proliferation. We also demonstrated that both Cpn60 and unusual mitochondrial ADP/ATP transporter (mitochondria carrier family, MCF) are important for the mitosome functions. Immunoelectron microscopy demonstrated that the enzymes involved in sulfate activation, Cpn60, and mitochondrial carrier family were differentially distributed within the electron dense, double membrane-bounded organelles. The importance and topology of the components in E. histolytica mitosomes reinforce the notion that they are not "rudimentary" or "residual" mitochondria, but represent a uniquely evolved crucial organelle in E. histolytica.
    PLoS Neglected Tropical Diseases 08/2011; 5(8):e1263. · 4.57 Impact Factor
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    ABSTRACT: To determine the mechanism of trifluoromethionine resistance in Entamoeba histolytica and evaluate the impact of acquired drug resistance on virulence. Trifluoromethionine-resistant amoebae were selected in vitro and examined for cross-resistance to antiamoebic drugs, stability of resistance, methionine γ-lyase (MGL) activity, cell adhesion and virulence. Targeted gene silencing was performed to confirm the role of EhMGL. Trophozoites with a resistance index of 154 were obtained. The cells were susceptible to chloroquine, metronidazole, paromomycin and tinidazole, but remained resistant to trifluoromethionine in the absence of drug pressure. A complete lack of EhMGL activity accompanied by increased adhesion and decreased cytolysis were also observed. Silencing of the EhMGL genes resulted in trifluoromethionine resistance. This study provides the first demonstration of trifluoromethionine resistance in a parasitic protozoon. Repression of gene expression of drug targets represents a novel mechanism of resistance in E. histolytica. The information obtained from this work should help further development of trifluoromethionine derivatives that have lower chances of inducing resistance.
    Journal of Antimicrobial Chemotherapy 06/2011; 66(9):2045-52. · 5.34 Impact Factor
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    ABSTRACT: Entamoeba histolytica, an enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. E. histolytica completely lacks glutathione metabolism but possesses L-cysteine as the principle low molecular weight thiol. L-Cysteine is essential for the structure, stability, and various protein functions, including catalysis, electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes. Recently, we demonstrated that in E. histolytica, L-cysteine regulates various metabolic pathways including energy, amino acid, and phospholipid metabolism. In this study, employing custom-made Affymetrix microarrays, we performed time course (3, 6, 12, 24, and 48 h) gene expression analysis upon L-cysteine deprivation. We identified that out of 9,327 genes represented on the array, 290 genes encoding proteins with functions in metabolism, signalling, DNA/RNA regulation, electron transport, stress response, membrane transport, vesicular trafficking/secretion, and cytoskeleton were differentially expressed (≥3 fold) at one or more time points upon L-cysteine deprivation. Approximately 60% of these modulated genes encoded proteins of no known function and annotated as hypothetical proteins. We also attempted further functional analysis of some of the most highly modulated genes by L-cysteine depletion. To our surprise, L-cysteine depletion caused only limited changes in the expression of genes involved in sulfur-containing amino acid metabolism and oxidative stress defense. In contrast, we observed significant changes in the expression of several genes encoding iron sulfur flavoproteins, a major facilitator super-family transporter, regulator of nonsense transcripts, NADPH-dependent oxido-reductase, short chain dehydrogenase, acetyltransferases, and various other genes involved in diverse cellular functions. This study represents the first genome-wide analysis of transcriptional changes induced by L-cysteine deprivation in protozoan parasites, and in eukaryotic organisms where L-cysteine represents the major intracellular thiol.
    BMC Genomics 05/2011; 12:275. · 4.40 Impact Factor
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    ABSTRACT: L-cysteine is ubiquitous in all living organisms and is involved in a variety of functions, including the synthesis of iron-sulfur clusters and glutathione and the regulation of the structure, stability, and catalysis of proteins. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, L-cysteine plays an essential role in proliferation, adherence, and defense against oxidative stress; however, the essentiality of this amino acid in the pathways it regulates is not well understood. In the present study, we applied capillary electrophoresis time-of-flight mass spectrometry to quantitate charged metabolites modulated in response to L-cysteine deprivation in E. histolytica, which was selected as a model for examining the biological roles of L-cysteine. L-cysteine deprivation had profound effects on glycolysis, amino acid, and phospholipid metabolism, with sharp decreases in the levels of L-cysteine, L-cystine, and S-adenosylmethionine and a dramatic accumulation of O-acetylserine and S-methylcysteine. We further demonstrated that S-methylcysteine is synthesized from methanethiol and O-acetylserine by cysteine synthase, which was previously considered to be involved in sulfur-assimilatory L-cysteine biosynthesis. In addition, L-cysteine depletion repressed glycolysis and energy generation, as it reduced acetyl-CoA, ethanol, and the major nucleotide di- and triphosphates, and led to the accumulation of glycolytic intermediates. Interestingly, L-cysteine depletion increased the synthesis of isopropanolamine and phosphatidylisopropanolamine, and it was confirmed that their increment was not a result of oxidative stress but was a specific response to L-cysteine depletion. We also identified a pathway in which isopropanolamine is synthesized from methylglyoxal via aminoacetone. To date, this study represents the first case where L-cysteine deprivation leads to drastic changes in core metabolic pathways, including energy, amino acid, and phospholipid metabolism.
    Journal of Biological Chemistry 10/2010; 285(50):39160-70. · 4.65 Impact Factor
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    ABSTRACT: We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.
    Journal of Biological Chemistry 08/2010; 285(35):26889-99. · 4.65 Impact Factor
  • International Journal of Antimicrobial Agents - INT J ANTIMICROBIAL AGENTS. 01/2010; 35(4):417-417.
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    ABSTRACT: The genome sequence of the enteric protozoan parasite Entamoeba histolytica suggests that amino acid catabolism plays an important role in energy metabolism. In the present study, we described kinetic and regulatory properties of catabolic l-threonine and l-serine dehydratase (TD) from E. histolytica. TD catalyses the pyridoxal phosphate-dependent dehydrative deamination of l-threonine and l-serine to ammonia and keto acids (2-oxobutyrate and pyruvate, respectively). E. histolytica possesses two TD isotypes (EhTD1-2) showing 38% mutual identity, a calculated molecular mass of 45.0 or 46.5kDa, and an isoelectric point of 6.68 or 5.88, respectively. Only EhTD1 showed l-threonine and l-serine dehydrative deaminating activities whereas EhTD2, in which the amino acid residues involved in the substrate and cofactor binding were not conserved, was devoid of these activities. The k(cat)/K(m) value of EhTD1 was >3 fold higher for l-threonine than l-serine. EhTD1 was inhibited by l-cysteine in a competitive manner with the K(i) values of 1.1mM and 2.2mM for l-serine and l-threonine, respectively. EhTD1 was insensitive to the allosteric activation by AMP or CMP. Three major substitutions of EhTD1 likely attribute to the insensitivity. EhTD1 was also inhibited about 50% by 20mM 2-oxobutyrate, pyruvate, and glyoxylate; the inhibition was not, however, reversed by AMP. Together, these data showed that EhTD1 possesses unique regulatory properties distinct from other organisms and may play an important role in energy metabolism via amino acid degradation in E. histolytica.
    Molecular and Biochemical Parasitology 11/2009; 170(2):100-4. · 2.73 Impact Factor
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    ABSTRACT: Amoebiasis, caused by infection with the enteric protist Entamoeba histolytica, is one of the major parasitic diseases. Although metronidazole and its derivatives are currently employed in therapy, the paucity of effective drugs and potential clinical resistance necessitate the development of a novel drug. Trifluoromethionine (TFM) is a promising lead compound for antiamoebic drugs. To potentiate the antiamoebic effect of TFM, we synthesised various amide derivatives of TFM and evaluated their cytotoxicity. The amide derivatives of TFM were observed to have a superior cytotoxic effect compared with TFM and metronidazole against E. histolytica in vitro. Although TFM showed cytotoxicity following degradation by methionine gamma-lyase, the derivatives were degraded by the enzyme less efficiently compared with TFM. We further demonstrated that a representative derivative was hydrolysed by the amoebic cell lysate to first yield TFM, followed by degradation similar to TFM. Hydrolysis was partially inhibited by protease inhibitors. A single subcutaneous or oral administration of TFM and its amide derivatives also effectively prevented the formation of amoebic liver abscess in a rodent model. These data demonstrate the improved effectiveness of TFM derivatives against E. histolytica infection and elucidate the mechanisms underlining the mode of action of these compounds.
    International journal of antimicrobial agents 11/2009; 35(1):56-61. · 3.03 Impact Factor
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    Dan Sato, Tomoyoshi Nozaki
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    ABSTRACT: Sulfur-containing amino acids (SAAs) are essential components in many biological processes and ubiquitously distributed to all organisms. Both biosynthetic and catabolic pathways of SAAs are heterogeneous among organisms and between developmental stages, and regulated by the environmental changes. Limited lineage of organisms ranging from archaea to plants, but not human, possess a unique enzyme methionine gamma-lyase (MGL, EC 4.4.1.11) to directly degrade SAA to alpha-keto acids, ammonia, and volatile thiols. The reaction mechanisms and the physiological roles of this enzyme are partially demonstrated by the enzymological analyzes, structure determination, isotopic labeling of the intermediate metabolites, and functional analyzes of deficient mutants. MGL has been exploited as a drug target for the infectious diseases caused by parasitic protozoa and anaerobic periodontal bacteria. In addition, MGL has been utilized to develop therapeutic interventions of various cancers, by introducing recombinant proteins to deplete methionine essential for the growth of cancer cells. In this review, we discuss the current understanding of enzymological properties, putative physiological roles, and therapeutic applications of MGL.
    International Union of Biochemistry and Molecular Biology Life 10/2009; 61(11):1019-28. · 2.79 Impact Factor
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    ABSTRACT: L-Methionine gamma-lyase (MGL) is a pyridoxal phosphate-dependent enzyme that is involved in the degradation of sulfur-containing amino acids. MGL is an attractive drug target against amoebiasis because the mammalian host of its causative agent Entamoeba histolytica lacks MGL. For the development of anti-amoebic agents based on the structure of MGL, one of two MGL isoenzymes (EhMGL1) was crystallized in the monoclinic space group P2(1), with unit-cell parameters a = 99.12, b = 85.38, c = 115.37 A, beta = 101.82 degrees . The crystals diffract to beyond 2.0 A resolution. The presence of a tetramer in the asymmetric unit (4 x 42.4 kDa) gives a Matthews coefficient of 2.8 A(3) Da(-1) and a solvent content of 56%. The structure was solved by the molecular-replacement method and structure refinement is now in progress.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2008; 64(Pt 8):697-9. · 0.55 Impact Factor

Publication Stats

187 Citations
86.73 Total Impact Points

Institutions

  • 2013
    • Tokyo Metropolitan University
      • Department of Chemistry
      Tokyo, Tokyo-to, Japan
  • 2008–2013
    • Keio University
      • Institute for Advanced Biosciences
      Tokyo, Tokyo-to, Japan
  • 2007–2012
    • National Institute of Infectious Diseases, Tokyo
      Edo, Tōkyō, Japan