Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen <i>Trypanosoma Cruzi</i>

Schöneck, Ralf; Billaut-Mulot, Odile; Numrich, Petra; Ouaissi, M. A.; Krauth-Siegel, R. Luke

doi:10.1111/j.1432-1033.1997.00739.x

Cited by 54 publications

(47 citation statements)

References 48 publications

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“…Isolation of Genomic DNA and Southern Blot-Genomic DNA from a culture of bloodstream TC221 T. brucei cells as well as from T. cruzi were prepared as described (25,26). 10 g of DNA was digested with StyI, and the fragments were separated on a 0.7% agarose gel.…”

Section: Methodsmentioning

confidence: 99%

Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei brucei

Reckenfelderbäumer

Lüdemann

Schmidt

et al. 2000

Journal of Biological Chemistry

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Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/ trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K m value of 6 M but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.

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Section: Methodsmentioning

confidence: 99%

Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei brucei

Reckenfelderbäumer

Lüdemann

Schmidt

et al. 2000

Journal of Biological Chemistry

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“…The very basic pI of peroxidase III is mainly due to its N-terminal extension. The sequence MLRSSRKKMSAA is rich in positively charged and hydroxylated residues and shows homology to other exceptionally short (7-9 amino acid residues) mitochondrial targeting signals of trypanosomatids (34,35). When the gene is overexpressed from the pQE-30/pxIII in E. coli, two protein species are obtained, one with the His 6 tag and the complete N-terminal sequence and a second about a 2-kDa smaller protein, which does not bind to the metal matrix.…”

Section: Analysis Of the Genomic Locus Of The Trypanosoma Bruceimentioning

confidence: 99%

A Second Class of Peroxidases Linked to the Trypanothione Metabolism

Hillebrand

Schmidt

Krauth‐Siegel

2003

Journal of Biological Chemistry

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Trypanosoma brucei, the causative agent of African sleeping sickness, has three nearly identical genes encoding cysteine homologues of classical selenocysteinecontaining glutathione peroxidases. The proteins are expressed in the mammalian and insect stages of the parasite. One of the genes, which contains a mitochondrial as well as a glycosomal targeting signal has been overexpressed. The recombinant T. brucei peroxidase has a high preference for the trypanothione/tryparedoxin couple as electron donor for the reduction of different hydroperoxides but accepts also T. brucei thioredoxin. The apparent rate constants k 2 for the regeneration of the reduced enzyme are 2 ؋ 10 5 M ؊1 s ؊1with tryparedoxin and 5 ؋ 10 3 M ؊1 s ؊1 with thioredoxin. No saturation kinetics was observed and the rate-limiting step of the overall reaction is reduction of the hydroperoxide. With glutathione, the peroxidase has marginal activity and reduction of the enzymes becomes limiting with a k 2 value of 3 M ؊1 s ؊1 . The T. brucei peroxidase, in contrast to the related Trypanosoma cruzi enzyme, also accepts hydrogen peroxide as substrate. The catalytic efficiency of the peroxidase studied here is comparable with that of the peroxiredoxin-like tryparedoxin peroxidases, which shows that trypanosomes possess two distinct peroxidase systems both dependent on the unique dithiol trypanothione.Parasitic trypanosomatids are the causative agents of lifethreatening tropical diseases such as American Chagas' disease (Trypanosoma cruzi), African sleeping sickness (T. brucei gambiense and T. b. rhodesiense), Nagana cattle disease (T. congolense, T. b. brucei), and the three manifestations of leishmaniasis. All these protozoa have in common that they possess an unparalleled thiol metabolism (1-3) where glutathionylspermidine conjugates are the main non-protein thiols. Trypanothione (T(SH) 2 ; N 1 ,N 8 -bis(glutathionyl)spermidine) (4) is kept in the dithiol state by the flavoenzyme trypanothione reductase, which catalyzes the NADPH-dependent reduction of trypanothione disulfide. Several genetic approaches revealed that trypanothione reductase is essential for the parasites (Ref. So far the trypanothione metabolism is the only known thiol system of the parasites where it replaces the otherwise ubiquitous glutathione/glutathione reductase couple. We could show that African trypanosomes contain also a classical thioredoxin but the respective specific reductase has not yet been described in any kinetoplastid organism (6). Trypanothione is the donor of reducing equivalents in several vital pathways of the parasites, like reduction dehydroascorbate (7), synthesis of deoxyribonucleotides by ribonucleotide reductase (8), conjugation and export of metals and drugs (9 -11), and the detoxication of hydroperoxides (12-14). Trypanosomes are exposed to various reactive oxygen species such as superoxide anions, hydrogen peroxide, and products of the host myeloperoxidase system. However, their ability to cope with oxidative stress seems to be surprisingly weak. Althou...

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“…The mRNA is about 0.8 kb larger than the gene. This is due to 82 nt at the 5' end which include the spliced leader sequence and a long 3' untranslated region [24]. The slightly higher mRNA level in the short stumpy forms does not seem to be significant, but it clearly shows that the mRNA is not down-regulated although these cells do not need deoxyribonucleotides for genome replication.…”

Section: Northern Blot Analysismentioning

confidence: 83%

Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

et al. 1997

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As a first step towards the understanding of the trypanosomal synthesis of deoxyribonucleotides we describe the cloning of the gene of ribonucleotide reductase subunit R2 from 72 brucei hrucei and its overexpression in E. coli. Materials and methods DNA and RNA preparation2;4 l08 long slender 72 brucei cells were suspended in 10 mM Tris-HC1, 250 mM NaCI, 0.5% Nonidet P-40, pH 8.0. After 5 rain incubation on ice the suspension was centrifuged and the cell pellet was homogenized in 200 lal 10 mM Tris-HC1, 10 mM NaC1, 10 mM EDTA, 0.5% SDS, pH 8.0. 50 lag proteinase K were added and the mixture was incubated for 1 h at 37°C. DNA was purified by phenol extraction.RNA was isolated from bloodstream long slender and short stumpy as well as procyclic stages of 72 brucei using the RNA Easy Kit (Qiagen) according to the instructions of the manufacturer.

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Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi

Cited by 54 publications

References 48 publications

Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei brucei

Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei brucei

A Second Class of Peroxidases Linked to the Trypanothione Metabolism

Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

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