The malaria parasite Plasmodium falciparum possesses a functional thioredoxin system

Krnajski, Zita; Gilberger, Tim‐Wolf; Walter, Rolf D.; Müller, Sylke

doi:10.1016/s0166-6851(00)00372-8

Cited by 45 publications

(40 citation statements)

References 56 publications

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“…Furthermore, it has been shown that TrxR/ Trx are components of an efficient antioxidant system in the cell (7,12,13). Despite these important roles, a functional thioredoxin redox system is dispensable in bacteria and yeast, and it was shown that there appears to be a high degree of redundancy for almost all proteins of the thioredoxin redox cascade, as long as there is a second system present in the cell that can compensate for the loss of function of the Trx system (50).…”

Section: Discussionmentioning

confidence: 99%

Thioredoxin Reductase Is Essential for the Survival ofPlasmodium falciparum Erythrocytic Stages

Krnajski¹,

Gilberger²,

Walter³

et al. 2002

Journal of Biological Chemistry

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102

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The human malaria parasite Plasmodium falciparum poses an increasing threat to human health in the tropical regions of the world, and the validation and assessment of possible drug targets is required for the development of new antimalarials. It has been shown that the erythrocytic stages of the parasites, which are responsible for the pathology of the disease in humans, are under enhanced oxidative stress and are particularly vulnerable to exogenous challenges by reactive oxygen species. Therefore it is postulated that the disruption of the antioxidant and/or redox systems of the parasite is a feasible way to interfere with their development during erythrocytic schizogony. In order to test this suggestion thioredoxin reductase (TrxR), an enzyme heavily involved in maintenance of redox homeostasis and antioxidant defense, was knocked out in P. falciparum. It was impossible to generate parasites with a disrupted trxR gene suggesting that TrxR is essential for P. falciparum erythrocytic stages. Technical problems were excluded by transfecting a 3 replacement construct, which recombined correctly and transfectants did not show any phenotypic alterations. In order to prove that the trxR knockout was responsible for the lethal phenotype of the null mutants, a co-transfection with both the knockout construct and a construct containing the trxR coding region under the control of the calmodulin promoter was conducted. Despite the disruption of the trxR gene, parasites were viable. In a Southern blot analysis a complicated restriction pattern was obtained, but it was shown by pulse field gel electrophoresis and field inverse gel electrophoreses that only the trxR gene locus on chromosome 9 was targeted by the constructs. It was found that the co-transfected constructs form concatemeric structures prior to integration into the trxR gene locus, which is further supported by plasmid rescue followed by restriction analyses of the plasmids. Northern and Western blot analyses proved that the co-transfectants highly overexpress TrxR from the introduced gene. Our results demonstrate that TrxR is essential for the survival of the erythrocytic stages of P. falciparum.

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Section: Discussionmentioning

confidence: 99%

Thioredoxin Reductase Is Essential for the Survival ofPlasmodium falciparum Erythrocytic Stages

Krnajski¹,

Gilberger²,

Walter³

et al. 2002

Journal of Biological Chemistry

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102

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“…Materials-Recombinant P. falciparum TrxR (PfTrxR) wildtype and PfTrxR variant proteins were generated as described in (19), and PfTrx was recombinantly expressed and purified as described in (35,36). Ni-NTA agarose was purchased from Qiagen.…”

Section: Methodsmentioning

confidence: 99%

Identification of Acid-Base Catalytic Residues of High-Mr Thioredoxin Reductase from Plasmodium falciparum

McMillan

Arscott

Ballou

et al. 2006

Journal of Biological Chemistry

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High-M r thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acidbase catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.

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“…The catalytic efficiency (k cat /K m ) for E. coli Trx was thus 100-fold lower than for HvTrxh1 and HvTrxh2, indicating that a noncognate Trx is not a good substrate for the NTR. Similarly, thioredoxin reductases from Caenorhabditis elegans (Lacey and Hondal, 2006) and Plasmodium falciparum (Krnajski et al, 2001) also show high activity toward E. coli Trx, but with a high K m . Kinetic parameters were also determined using Arabidopsis NTR (AtNTR; Jacquot et al, 1994), which had lower affinity and lower activity for barley Trx h isoforms compared with barley NTRs (Table I).…”

Section: Enzyme Activitymentioning

confidence: 99%

The NADPH-Dependent Thioredoxin Reductase/Thioredoxin System in Germinating Barley Seeds: Gene Expression, Protein Profiles, and Interactions between Isoforms of Thioredoxin h and Thioredoxin Reductase

2007

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The NADPH-dependent thioredoxin reductase (NTR)/thioredoxin (Trx) system catalyzes disulfide bond reduction in the cytoplasm and mitochondrion. Trx h is suggested to play an important role in seed development, germination, and seedling growth. Plants have multiple isoforms of Trx h and NTR; however, little is known about the roles of the individual isoforms. Trx h isoforms from barley (Hordeum vulgare) seeds (HvTrxh1 and HvTrxh2) were characterized previously. In this study, two NTR isoforms (HvNTR1 and HvNTR2) were identified, enabling comparison of gene expression, protein appearance, and interaction between individual NTR and Trx h isoforms in barley embryo and aleurone layers. Although mRNA encoding both Trx h isoforms is present in embryo and aleurone layers, the corresponding proteins differed in spatiotemporal appearance. HvNTR2, but not HvNTR1, gene expression seems to be regulated by gibberellic acid. Recombinant HvNTR1 and HvNTR2 exhibited virtually the same affinity toward HvTrxh1 and HvTrxh2, whereas HvNTR2 has slightly higher catalytic activity than HvNTR1 with both Trx h isoforms, and HvNTR1 has slightly higher catalytic activity toward HvTrxh1 than HvTrxh2. Notably, both NTRs reduced Trx h at the acidic conditions residing in the starchy endosperm during germination. Interspecies reactions between the barley proteins and Escherichia coli Trx or Arabidopsis thaliana NTR, respectively, occurred with 20- to 90-fold weaker affinity. This first investigation of regulation and interactions between members of the NTR/Trx system in barley seed tissues suggests that different isoforms are differentially regulated but may have overlapping roles, with HvNTR2 and HvTrxh1 being the predominant isoforms in the aleurone layer.

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The malaria parasite Plasmodium falciparum possesses a functional thioredoxin system

Cited by 45 publications

References 56 publications

Thioredoxin Reductase Is Essential for the Survival ofPlasmodium falciparum Erythrocytic Stages

Thioredoxin Reductase Is Essential for the Survival ofPlasmodium falciparum Erythrocytic Stages

Identification of Acid-Base Catalytic Residues of High-Mr Thioredoxin Reductase from Plasmodium falciparum

The NADPH-Dependent Thioredoxin Reductase/Thioredoxin System in Germinating Barley Seeds: Gene Expression, Protein Profiles, and Interactions between Isoforms of Thioredoxin h and Thioredoxin Reductase

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