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Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica*

  • Ghulam Jeelani
    Footnotes
    Affiliations
    Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

    Center for Integrated Medical Research, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan
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  • Afzal Husain
    Affiliations
    Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
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  • Dan Sato
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan
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  • Vahab Ali
    Affiliations
    Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna-800007, India
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  • Makoto Suematsu
    Affiliations
    Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan
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  • Tomoyoshi Soga
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan
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  • Tomoyoshi Nozaki
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-3-5285-1111 (ext. 2600); Fax.: 81-3-5285-1219
    Footnotes
    Affiliations
    Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
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  • Author Footnotes
    * This work was supported by Grants-in-aid for Scientific Research 18GS0314, 18050006, and 18073001 (to T. N.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, a grant for research on emerging and re-emerging infectious diseases from the Ministry of Health, Labour, and Welfare of Japan (H20-Shinkosaiko-016), and a grant for research to promote the development of anti-AIDS pharmaceuticals from the Japan Health Sciences Foundation (to T. N.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.
    1 Supported in part by the Global Center of Excellence Program for Human Metabolomic System Biology of the Ministry of Education Culture, Sports, Science, and Technology.
Open AccessPublished:June 30, 2010DOI:https://doi.org/10.1074/jbc.M110.106310
      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 Km and higher kcat/Km 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.

      Introduction

      Glutamate synthase (glutamine:2-oxoglutarate amidotransferase, GOGAT
      The abbreviations used are: GOGAT
      glutamine:2-oxoglutarate amidotransferase
      EhNO
      E. histolytica NADPH-dependent oxidoreductase
      rEhNO
      recombinant EhNO
      INT
      iodonitrotetrazolium
      CE
      capillary electrophoresis
      SoFd
      S. oleracea ferredoxin
      EhFd1
      E. histolytica ferredoxin.
      ) is an iron sulfur flavoprotein that catalyzes the transfer of the amide group of l-glutamine to 2-oxoglutrate to yield l-glutamate and is a key enzyme in the nitrogen assimilation pathway. In eubacteria, this enzyme is dependent on the pyridine nucleotide NAD(P)H for its reducing equivalents and is composed of large 150-kDa (α) and small 50-kDa (β) subunits that together form the active αβ protomer (
      • Ratti S.
      • Curti B.
      • Zanetti G.
      • Galli E.
      ). The structural genes encoding the α and β subunit polypeptides are commonly designated gltB and gltD, respectively, and lie adjacent on the chromosome with the α subunit preceding the β subunit, except in γ-proteobacteria, where the gene order is reversed. The small subunit of eubacterial glutamate synthase shows sequence similarity to several other protein domains and enzyme subunits (
      • Rosenbaum K.
      • Jahnke K.
      • Curti B.
      • Hagen W.R.
      • Schnackerz K.D.
      • Vanoni M.A.
      ,
      • Tóth A.
      • Takács M.
      • Groma G.
      • Rákhely G.
      • Kovács K.L.
      ) and is, therefore, proposed to represent a prototype domain used in many different cellular processes to transfer electrons from NAD(P)H to an acceptor protein or protein domain of unknown function (
      • Vanoni M.A.
      • Curti B.
      ). In concord with this view, numerous organisms have been recently identified to possess glutamate synthase β subunit-like genes based on DNA sequence homology (
      • Vanoni M.A.
      • Curti B.
      ,
      • Stutz H.E.
      • Reid S.J.
      ); however, the organisms often lack a gene encoding the corresponding α subunit, or the β subunit is not present adjacent to the α subunit and is, therefore, transcribed independently (
      • Stutz H.E.
      • Reid S.J.
      ,
      • Saum S.H.
      • Sydow J.F.
      • Palm P.
      • Pfeiffer F.
      • Oesterhelt D.
      • Müller V.
      ). To our knowledge, among the organisms lacking a putative GOGAT α subunit, only the GOGAT β subunit from Thermococcus kodakaraensis (renamed from Pyrococcus sp. KOD1) has been functionally associated with independent GOGAT activity (
      • Jongsareejit B.
      • Rahman R.N.
      • Fujiwara S.
      • Imanaka T.
      ).
      Entamoeba histolytica, the causative agent of human amebiasis, is an enteric protozoan parasite responsible for amebic colitis and extraintestinal abscesses in approximately 50 million inhabitants of endemic areas (
      • World Health Organization
      WHO/PAHO/UNESCO Report: A consultation with experts on amebiasis. Mexico City, Mexico 28–29 January, 1997.
      ). As is the case with other microaerophilic parasitic infections, such as giardiasis and trichomoniasis, the 5-nitroimidazole drug metronidazole has been established as the most effective treatment of amebiasis. Because of the high prevalence of these infections (
      • Upcroft P.
      • Upcroft J.A.
      ) and because of its role as a second-line defense against Helicobacter pylori infections (
      • Hoffman J.S.
      • Cave D.R.
      ), metronidazole has been included in the list of “essential medicines” by the World Health Organization (
      • World Health Organization
      WHO Model List of Essential Medicines.
      ). Metronidazole is a prodrug that requires reduction of the nitro group to generate the cytotoxic nitroradical anion that undergoes further reduction resulting in the generation of nitrosoimidazole (
      • Müller M.
      ,
      • Moreno S.N.
      • Docampo R.
      ). This active form can then react with sulfhydryl groups (
      • West S.B.
      • Wislocki P.G.
      • Fiorentini K.M.
      • Alvaro R.
      • Wolf F.J.
      • Lu A.Y.
      ) and DNA (
      • Ludlum D.B.
      • Colinas R.J.
      • Kirk M.C.
      • Mehta J.R.
      ) while being further reduced to an amine via a hydroxylamine intermediate. Here, we report for the first time multiple novel roles of two GOGAT β subunit-like proteins in E. histolytica. We demonstrated that they are not associated with glutamate synthase activity but instead exhibit robust reductase activities against l-cystine, ferredoxin, and ferric ion and are also involved in the response to oxidative stress. In addition, we showed that these enzymes can be capable of reducing and activating metronidazole and, thus, are responsible for its observed toxicity against E. histolytica. We designated the novel NADPH-dependent oxidoreductases as EhNO1 and -2.

      DISCUSSION

      In the present study we demonstrated novel enzymatic reactions catalyzed by a new class of FAD- and 2[4Fe-4S]-containing NADPH-dependent oxidoreductases from E. histolytica, which had been initially discovered by virtue of tightly regulated gene expression in correlation with l-cysteine concentrations. Although the two EhNOs characterized in this study had been annotated before this study based on their high degree of homology with GOGAT β subunit and β subunit-like genes from a variety of organisms, their biochemical function was unknown. The fact that the two EhNOs shared significant similarity with homologs from archaeal organisms raised the question of whether they represented a prototype GOGAT protein, similar to the β subunit protein from Pyrococcus, which was reported to possess NADPH-dependent GOGAT activity and be capable of both glutamine and ammonia-dependent synthesis in the absence of the α subunit (
      • Jongsareejit B.
      • Rahman R.N.
      • Fujiwara S.
      • Imanaka T.
      ). However, we were unable to observe glutamate synthase activity of the EhNOs under similar conditions used for the Pyrococcus glutamate synthase. Furthermore, the expression of the GOGAT β subunit failed to restore glutamate auxotrophy in an E. coli GOGAT α subunit-deficient strain (
      • Stutz H.E.
      • Reid S.J.
      ,
      • Deane S.M.
      • Rawlings D.E.
      ). In addition, it was somewhat puzzling how the Pyrococcus GOGAT β subunit functioned in substrate binding and catalysis without the α subunit, which has been shown to be responsible for substrate recognition (
      • Vanoni M.A.
      • Fischer F.
      • Ravasio S.
      • Verzotti E.
      • Edmondson D.E.
      • Hagen W.R.
      • Zanetti G.
      • Curti B.
      ). Thus, it was thought that the EhNO β subunit-like proteins may be involved in reactions other than glutamate synthesis.
      The physiological roles of EhNO1 and EhNO2 have not been unequivocally demonstrated because our attempt to repress EhNO expression by gene silencing (
      • Bracha R.
      • Nuchamowitz Y.
      • Anbar M.
      • Mirelman D.
      ) failed (data not shown), and gene knock-out has not been accomplished in E. histolytica. Nevertheless, our present enzymological characterization revealed the physiological significance of the presence of the two isotypes of EhNOs. EhNO2 appears to play an important role in the reduction of cystine to l-cysteine. Because l-cysteine is partially present in the oxidized form inside cells (CE-MS analysis, Fig. 2B), l-cystine reduction is necessary before the utilization of l-cysteine, which has been implicated in the attachment to matrix, elongation, motility, growth, and anti-oxidative defense (
      • Nozaki T.
      • Asai T.
      • Sanchez L.B.
      • Kobayashi S.
      • Nakazawa M.
      • Takeuchi T.
      ,
      • Gillin F.D.
      • Diamond L.S.
      ,
      • Gillin F.D.
      • Diamond L.S.
      ). Transcriptomic analysis demonstrated that the transcription of EhNO2, but not EhNO1, is tightly regulated by extracellular l-cysteine concentrations. Furthermore, the measured kinetic parameters indicate that EhNO2 possesses 4-fold higher l-cystine reduction efficiency than EhNO1.
      The acquisition of iron and subsequent assimilation into cellular proteins are ubiquitously essential for life. However, at physiological pH under aerobic conditions, iron is present as Fe3+ hydroxides and oxyhydroxides or in a complex with ferric-specific chelators, e.g. siderophores (
      • Barchini E.
      • Cowart R.E.
      ). Subsequent reduction of complexed Fe3+ is accomplished by ferric reductases using NAD(P)H as the electron donor (
      • Lesuisse E.
      • Crichton R.R.
      • Labbe P.
      ), with the resulting Fe2+ being subsequently released and incorporated into iron-containing proteins (
      • Guerinot M.L.
      ). We showed that rEhNO1 catalyzes the reduction of ferric ion >100-fold more efficiently than rEhNO2 (Table 2), suggesting that EhNO1 is mainly involved in ferric reduction. We also confirmed that EhNO1 functions as a ferredoxin:NADP+ reductase, similar to the recently reported ferric reductase from Pseudomonas putida (
      • Yeom J.
      • Jeon C.O.
      • Madsen E.L.
      • Park W.
      ), by catalyzing reversible electron transfer between one molecule of NADP+/NADPH and two molecules of ferredoxin. In vitro cross-linking of the two EhNOs with ferredoxin indicate that only EhNO1 forms a stable complex with E. histolytica ferredoxin (EhFd1), whereas both EhNO1 and EhNO2 physically interact with spinach ferredoxin (Fig. 4B), indicating that the specificity toward ferredoxin differs between these two proteins. The E. histolytica genome encodes four types of ferredoxins which are highly divergent at the primary sequence level and also in the Fe-S clusters. We, therefore, hypothesize that EhNO2 interacts with a ferredoxin(s) other than EhFd1 in E. histolytica, a speculation that is supported by the observed differential binding of photosynthetic and non-photosynthetic maize ferredoxins to root Zea mays ferredoxin:NADP+ reductase (
      • Onda Y.
      • Matsumura T.
      • Kimata-Ariga Y.
      • Sakakibara H.
      • Sugiyama T.
      • Hase T.
      ).
      E. histolytica is anaerobic/microaerophilic and possesses highly degenerated mitochondria that are incapable of oxidative phosphorylation and ATP generation. A crucial step in energy production via glycolysis and fermentation in E. histolytica involves the decarboxylation of pyruvate to acetyl CoA that is catalyzed by pyruvate:ferredoxin oxidoreductase (
      • Kerscher L.
      • Oesterhelt D.
      ). Concomitant with the decarboxylation of pyruvate, an electron is transferred to oxidized ferredoxin. Generally, reduced ferredoxin subsequently donates an electron to NAD(P) by the action of ferredoxin:NADP reductase, which serves to regenerate the intracellular pools of NAD(P)H and oxidized ferredoxin. However, as the Entamoeba genome does not contain an ferredoxin:NADP reductase homolog, it was unclear how NAD(P)H was regenerated. Our enzymological study indicates that EhNOs, and EhNO1 in particular, function as ferredoxin:NADP reductases and are involved in the regeneration of NADPH and oxidized ferredoxin required for continuous energy production.
      As stated above, E. histolytica possesses highly divergent mitochondria (
      • Tovar J.
      • Fischer A.
      • Clark C.G.
      ) and lacks a functional tricarboxylic acid cycle, cytochromes, and a conventional respiratory electron transport chain terminating in the reduction of oxygen to water. However, amebae can still tolerate up to 5% of oxygen in the gas phase (
      • Band R.N.
      • Cirrito H.
      ,
      • Reeves R.E.
      ) and consume oxygen (
      • Weinbach E.C.
      • Diamond L.S.
      ). As shown here, EhNOs are flavoproteins containing 1 mol of FAD as a prosthetic group per mol of enzyme. During the NADPH:flavin oxidoreductase reaction, NADPH binds to EhNOs, and two electrons are transferred to FAD to yield FADH2, which is immediately dissociated from the enzyme (
      • Inouye S.
      ). Under aerobic conditions, FADH2 is rapidly oxidized by molecular oxygen to yield H2O2 and FAD (
      • Gibson Q.H.
      • Hastings J.W.
      ). As E. histolytica amebae do not produce detectable amounts of H2O2 (
      • Lo H.
      • Reeves R.E.
      ), it is possible that H2O2 is further converted to water by peroxiredoxin (
      • Bruchhaus I.
      • Richter S.
      • Tannich E.
      ) and rubrerythrin (
      • Maralikova B.
      • Ali V.
      • Nakada-Tsukui K.
      • Nozaki T.
      • van der Giezen M.
      • Henze K.
      • Tovar J.
      ) to overcome oxidative stress. Under anaerobic conditions, EhNO1 catalyzes ferric ion reduction, whereas EhNO2 catalyzes cystine reduction. Based on the demonstrated reactions catalyzed by the two EhNOs, we have proposed functional roles for these two proteins in E. histolytica that are summarized in Fig. 6.
      Figure thumbnail gr6
      FIGURE 6Proposed in vivo reactions catalyzed by EhNO1 and -2. PFOR, pyruvate:ferredoxin oxidoreductase; Fd-red and Fd-oxi, reduced and oxidized form of ferredoxin; Prx, peroxiredoxin; Rbr, rubrerythrin; RNO2/RNO2°, metronidazole/reduced metronidazole.
      Metronidazole is a prodrug currently used to treat a number of microbial infections, and its activation requires intracellular reduction to produce cytotoxic short-lived radicals and other reactive species (
      • Goldman P.
      • Koch R.L.
      • Yeung T.C.
      • Chrystal E.J.
      • Beaulieu Jr., B.B.
      • McLafferty M.A.
      • Sudlow G.
      ). Entamoeba electron transport proteins, which have been reported to provide the source of electrons for the reductive activation of metronidazole, include ferredoxin (
      • Müller M.
      ), thioredoxin reductase (
      • Leitsch D.
      • Kolarich D.
      • Wilson I.B.H.
      • Altmann F.
      • Duchene M.
      ), and nitroreductase (
      • Pal D.
      • Banerjee S.
      • Cui J.
      • Schwartz A.
      • Ghosh S.K.
      • Samuelson J.
      ). We demonstrated that both EhNO1 and -2 catalyze metronidazole reduction in vitro (Table 1), and their overexpression confers increased sensitivity to this drug (Fig. 5B). This finding suggests that in addition to ferredoxin (
      • Müller M.
      ), pyruvate:ferredoxin oxidoreductase (
      • Müller M.
      ), thioredoxin (
      • Leitsch D.
      • Kolarich D.
      • Wilson I.B.H.
      • Altmann F.
      • Duchene M.
      ), and nitroreductase (
      • Pal D.
      • Banerjee S.
      • Cui J.
      • Schwartz A.
      • Ghosh S.K.
      • Samuelson J.
      ), EhNO1 and -2 are also involved in metronidazole activation in E. histolytica.
      In conclusion, we have demonstrated for the first time that two novel NADPH-dependent GOGAT small subunit-like proteins of E. histolytica function, at least in vitro, as cystine/ferric/ferredoxin:NADP+ reductase. We propose that they play a role in maintaining intracellular redox potential and may be responsible for metronidazole activation in this parasite. The physiological substrates and biological roles of the majority of oxidoreductases discovered by genome mining remain largely unknown. Vigorous attempts to discover the substrates and functions of individual oxidoreductases should unveil novel cellular metabolic processes in pathogens and cancer cells that may lead to the development of new chemotherapeutics.

      Acknowledgments

      We thank Kumiko Nakada-Tsukui, Fumika Mi-ichi, Takashi Makiuchi, and all other members of our laboratory for technical assistance and valuable discussions.

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