Thioredoxin-2 but Not Thioredoxin-1 Is a Substrate of Thioredoxin Peroxidase-1 from Drosophila melanogaster

Abstract

As Drosophila melanogaster does not contain glutathione reductase, the thioredoxin system has a key function for glutathione disulfide reduction in insects (Kanzok, S. M., Fechner, A., Bauer, H., Ulschmid, J. K., Mü ller, H. M., Botella-Munoz, J., Schneuwly, S., Schirmer, R. H., and Becker, K. (2001) Science 291, 643-646). In view of these unique conditions, the protein systems participating in peroxide metabolism and in redox signaling are of special interest. The genes for a second thioredoxin (DmTrx-2) and a thioredoxin peroxidase (DmTPx-1) were cloned and expressed, and the proteins were characterized. In its disulfide form, the 13-kDa protein thioredoxin-2 is a substrate of thioredoxin reductase-1 (K m ‫؍‬ 5.2 M, k cat ‫؍‬ 14.5 s ؊1 ) and in its dithiol form, an electron donor for TPx-1 (K m ‫؍‬ 9 M, k cat ‫؍‬ 5.4 s ؊1 ). DmTrx-2 is capable of reducing glutathione disulfide with a second order rate constant of 170 M ؊1 s ؊1 at pH 7.4 and 25°C. Western blot analysis indicated that this thioredoxin represents up to 1% of the extractable protein of D. melanogaster Schneider cells or whole fruit flies. Recombinant thioredoxin peroxidase-1 (subunit molecular mass ‫؍‬ 23 kDa) was found to be a decameric protein that can efficiently use Trx-2 but not Trx-1 as a reducing substrate. The new electron pathway found in D. melanogaster is also representative for insects that serve as vectors of disease. As a first step we have cloned and functionally expressed the gene that is the orthologue of DmTrx-2 in the malaria mosquito Anopheles gambiae.Aerobic life conditions that are based on metabolizing molecular oxygen are linked inseparably to the occurrence of reactive oxygen species. These compounds are cytotoxic for the organism but even more so for invaders such as microorganisms and tumor cells. The oxidative challenge is counteracted by a shield of enzymatic and non-enzymatic defense systems including superoxide dismutase (2 O 2

The Thioredoxin System of the Filamentous Fungus Aspergillus nidulans

“…With the thioredoxindependent peroxidase activity assay we showed that the PRX5-like protein reduces H 2 O 2 only in the presence of a functional thioredoxin system. Furthermore, the kinetic parameters of this enzyme for H 2 O 2 are in agreement with activities of a human thioredoxin peroxidase, a thioredoxin peroxidase from Plasmodium falciparum, and a thioredoxin peroxidase-1 from Drosophila melanogaster (52)(53)(54). This led us to conclude that the identified PRX5-like protein is a functional thioredoxin-dependent peroxidase in A. nidulans.…”

Microarray analysis of a pyrethroid resistant African malaria vector, Anopheles funestus, from southern Africa

“…It is possible that these 21 additional differentially expressed genes are unrelated to the resistance phenotype. It is interesting to note that several of them are putatively involved in the oxidative stress response (superoxide dismutases (SOD) [33; 34; 35], thioredoxins (TRX) [36], glutathione-S-transferases (GSTs) [37; 38; 39].…”

Active sites of thioredoxin reductases: Why selenoproteins?

“…All standard assays were carried out at 25°C. Recombinant N-terminally His-tagged DmTrx-2 was prepared as described (18). NADPH was from Biomol (Hamburg, Germany).…”

“…Concentrations of Cys-Sec mutants were determined from the selenium concentration as measured by atomic absorption spectroscopy (see Table 1). The concentration of oxidized DmTrx-2, purified as described earlier (18), was measured enzymatically by end-point determination by using excess NADPH and 1 unit͞ml of wild-type DmTrxR.…”