Reduction of dehydroascorbate by trypanothione

Rl, Krauth-Siegel; Lüdemann, Heike

doi:10.1016/0166-6851(96)02689-8

Cited by 53 publications

(31 citation statements)

References 22 publications

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“…Moreover, this activity could be reconstituted in the presence of trypanothione and its associated reductase when cell extracts were omitted from the assay, which suggests that DHA reduction occurs at the expense of T[SH] 2 by a nonenzymatic process (Fig. 2), in agreement with earlier studies (26).…”

Section: Resultssupporting

confidence: 78%

“…In most organisms ascorbate regeneration can occur nonenzymatically by, for example, interaction with glutathione, or by means of the activity of enzymes including protein disulfide isomerase and DHA reductase (44,45). In trypanosomatids both enzymatic and nonenzymatic mechanisms have been postulated for this recycling process (16,26). We were able to confirm the findings of Krauth-Siegel and Ludemann (26) regarding the nonenzymatic mechanism of this reaction in T. cruzi (Fig.…”

Section: Discussionsupporting

confidence: 81%

“…Both enzymatic and nonenzymatic mechanisms for the regeneration of ascorbate from its oxidized DHA form have been postulated in T. cruzi (16,26). To address this issue, the soluble fraction of dialyzed T. cruzi cell extracts was assayed for DHA reductase activity by monitoring the oxidation of NADPH to NADP ϩ (data not shown).…”

Section: Resultsmentioning

confidence: 99%

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Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Wilkinson

Obado

Maurício

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

133

116

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In most aerobic organisms hemoperoxidases play a major role in H 2O2-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis-Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H 2O2. To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.

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Section: Resultssupporting

confidence: 78%

Section: Discussionsupporting

confidence: 81%

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Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Wilkinson

Obado

Maurício

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

133

116

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“…Trypanosomes contain significant levels of ascorbate (20), and regeneration from its oxidized dehydroascorbate form occurs by a nonenzymatic reaction involving trypanothione (6,21). However, the role of ascorbate in trypanosomatid biology is poorly understood.…”

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confidence: 99%

Vitamin C biosynthesis in trypanosomes: A role for the glycosome

Wilkinson

Prathalingam

Taylor

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The capacity to synthesize vitamin C (ascorbate) is widespread in eukaryotes but is absent from humans. The last step in the biosynthetic pathway involves the conversion of an aldonolactone substrate to ascorbate, a reaction catalyzed by members of an FAD-dependent family of oxidoreductases. Here we demonstrate that both the African trypanosome, Trypanosoma brucei, and the American trypanosome, Trypanosoma cruzi, have the capacity to synthesize vitamin C and show that this reaction occurs in a unique single-membrane organelle, the glycosome. The corresponding T. brucei flavoprotein (TbALO) obeys Michaelis-Menten kinetics and can utilize both L-galactono-␥-lactone and D-arabinono-␥-lactone as substrate, properties characteristic of plant and fungal enzymes. We could detect no activity toward the mammalian enzyme substrate L-gulono-␥-lactone. TbALO null mutants (bloodstream form) were found to display a transient growth defect, a trait that was enhanced when they were cultured in medium in which the essential serum component had been pretreated with ascorbate oxidase to deplete vitamin C. It is implicit, therefore, that bloodstream-form trypanosomes also possess a capacity for ascorbate transport.ascorbic acid ͉ FAD ͉ oxidase ͉ Trypanosoma brucei

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“…The pivotal role of trypanothione in the antioxidant defense mechanisms of the parasites is well established (11,(13)(14)(15)(16)(17). As shown here, trypanothione is also involved in the parasite synthesis of DNA precursors (Fig.…”

Section: Discussionmentioning

confidence: 82%

Trypanothione-dependent Synthesis of Deoxyribonucleotides by Trypanosoma brucei Ribonucleotide Reductase

Dormeyer¹,

Reckenfelderbäumer

Lüdemann

et al. 2001

Journal of Biological Chemistry

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113

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Trypanosoma brucei, the causative agent of African sleeping sickness, synthesizes deoxyribonucleotides via a classical eukaryotic class I ribonucleotide reductase. The unique thiol metabolism of trypanosomatids in which the nearly ubiquitous glutathione reductase is replaced by a trypanothione reductase prompted us to study the nature of thiols providing reducing equivalents for the parasite synthesis of DNA precursors. Here we show that the dithiol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione, is a direct reductant of T. brucei ribonucleotide reductase with a K m value of 2 mM. This is the first example of a natural low molecular mass thiol directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryparedoxin, a 16-kDa parasite protein with a WCPPC active site motif. The K m value of T. brucei ribonucleotide reductase for T. brucei tryparedoxin is about 4 M. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regulation of deoxyribonucleotide synthesis by the redox state of the cell. The trypanothione/tryparedoxin system is a new system providing electrons for a class I ribonucleotide reductase, in addition to the well known thioredoxin and glutaredoxin systems described in other organisms.Ribonucleotide reductases (E.C. 1.17.4.1) catalyze the ratelimiting step in the de novo synthesis of DNA precursors and thus are key enzymes for the replication of an organism (1). African trypanosomes possess a typical eukaryotic class I ribonucleotide reductase (2-4). The genes encoding the Trypanosoma brucei R1 and R2 1 proteins have been cloned and overexpressed in Escherichia coli. Class I enzymes are tetrameric proteins composed of two R1 and R2 molecules each. The large R1 protein harbors the active site, as well as regulatory sites, and the small R2 protein contains a -oxo-bridged diiron cluster, which represents the Fe(III)-O 2Ϫ -Fe(III) cofactor in all eukaryotic ribonucleotide reductases, and a tyrosyl radical essential for catalysis (1, 5). T. brucei ribonucleotide reductase is regulated via the R2 subunit. Whereas the R1 protein is present throughout the life cycle of the parasite, the R2 protein is not found in cell cycle-arrested short stumpy trypanosomes (6

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Reduction of dehydroascorbate by trypanothione

Cited by 53 publications

References 22 publications

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Vitamin C biosynthesis in trypanosomes: A role for the glycosome

Trypanothione-dependent Synthesis of Deoxyribonucleotides by Trypanosoma brucei Ribonucleotide Reductase

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