Kinetic characterization of wild-type and mutant human thioredoxin glutathione reductase defines its reaction and regulatory mechanisms.

In most cells, the thioredoxin (Trx) and glutathione systems are essential in maintaining redox homeostasis. The selenoprotein thioredoxin glutathione reductase (TGR) is a hybrid enzyme in which a glutaredoxin (Grx) domain is linked to a thioredoxin reductase (TrxR). Notably, the protein is also capable of reducing glutathione disulfide (GSSG), thus representing an important link between the two redox systems. In this study, we recombinantly produced human TGR (hTGR wild-type) by fusing its open reading frame with a bacterial selenocysteine insertion sequence element and co-expressing the construct in Escherichia coli together with the selA, selB, and selC genes. Additionally, the Sec→Cys mutant (hTGRU642C ) of the full-length protein, the isolated TrxR domain (hTGR151-643 ) and the Grx domain containing a monothiol active site (hTGR1-150 ) were produced and purified. All four proteins were kinetically characterized in direct comparison using Trx, DTNB, HED, or GSSG as the oxidizing substrate. Interestingly, the HED reduction activity was Sec independent and comparable in the full-length protein and the isolated Grx domain, whereas the TrxR and glutathione reductase reactions were clearly selenocysteine dependent, with the GR reaction requiring the Grx domain. Site-directed mutagenesis studies revealed novel insights into the mechanism of GSSG reduction. Furthermore, we identified several glutathionylation sites in hTGR, including Cys93, Cys133, and Cys619, and an inhibitory effect of these modifications on enzyme activity. In contrast to other TGRs, for example, from platyhelminth parasites, hTGR did not exhibit hysteretic behavior. These findings provide new insights into the reaction mechanism and regulation of monothiol Grx-containing TGRs. DATABASE: EC numbers: 1.8.1.9; 1.8.1.B1.

Insight into the selenoproteome of the malaria parasite Plasmodium falciparum.

AIMS: The malaria parasite Plasmodium falciparum possesses four unique selenoproteins (PfSel1-PfSel4) which are likely to represent important components of the redox-regulatory network of this infectious agent. So far these proteins have only been characterized in silico. The aim of the present study was to gain further insight into the structural, biochemical, and functional properties of P. falciparum selenoproteins. RESULTS: Using (75)Se labeling in P. falciparum cell culture, the presence of selenoproteins in the parasite could be verified for the first time. Bioinformatic analyses indicated distant relatedness between the Plasmodium proteins and selenoproteins described in other organisms, namely between PfSel1 and SelK, PfSel2 and SelT, and between PfSel4 and SelS. For PfSel3 no remarkable similarities with proteins from other organisms were identified. All four proteins were recombinantly produced in Escherichia coli as UGA→UGU (selenocysteine→cysteine) mutants. Using green fluorescent protein (GFP)-fusion proteins and immunofluorescence, the subcellular localization of the four selenoprotein mutants was studied. PfSel1, PfSel2, and PfSel4 localized to the endoplasmic reticulum whereas PfSel3 was visualized in the nucleus and/or the apicoplast. Functional assays support the roles of PfSel1 and PfSel4 in cellular redox reactions. Transcriptional profiles of the four selenoproteins, and proteins involved in selenoprotein biosynthesis, indicate that their expression is regulated via the availability of selenium and via oxidative and nitrosative stress. INNOVATION: In this study the presence of selenoproteins in Plasmodium has been proven for the first time; the subcellular localization of the proteins and their relatedness to known selenoproteins have been systematically studied, and recombinant proteins as well as information on regulation of transcript levels have been obtained. CONCLUSION: Taken together, our data enhance our understanding of the functional role of selenoproteins in Plasmodium.

Comparative structural analysis of oxidized and reduced thioredoxin from Drosophila melanogaster.

Thioredoxins (Trx) participate in essential antioxidant and redox-regulatory processes via a pair of conserved cysteine residues. In dipteran insects like Drosophila and Anopheles, which lack a genuine glutathione reductase (GR), thioredoxins fuel the glutathione system with reducing equivalents. Thus, characterizing Trxs from these organisms contributes to our understanding of redox control in GR-free systems and provides information on novel targets for insect control. Cytosolic Trx of Drosophila melanogaster (DmTrx) is the first thioredoxin that was crystallized for X-ray diffraction analysis in the reduced and in the oxidized form. Comparison of the resulting structures shows rearrangements in the active-site regions. Formation of the C32-C35 disulfide bridge leads to a rotation of the side-chain of C32 away from C35 in the reduced form. This is similar to the situation in human Trx and Trx m from spinach chloroplasts but differs from Escherichia coli Trx, where it is C35 that moves upon change of the redox state. In all four crystal forms that were analysed, DmTrx molecules are engaged in a non-covalent dimer interaction. However, as demonstrated by gel-filtration analyses, DmTrx does not dimerize under quasi in vivo conditions and there is no redox control of a putative monomer/dimer equilibrium. The dimer dissociation constants K(d) were found to be 2.2mM for reduced DmTrx and above 10mM for oxidized DmTrx as well as for the protein in the presence of reduced glutathione. In human Trx, oxidative dimerization has been demonstrated in vitro. Therefore, this finding may indicate a difference in redox control of GR-free and GR-containing organisms.