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Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione.

ABSTRACT: Platyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate the existence of functional linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of Echinococcus granulosus, the platyhelminth responsible for hydatid disease. The glutathione reductase (GR) activity of TGR exhibited hysteretic behavior regulated by the [GSSG]/[GSH] ratio. This behavior was associated with glutathionylation by GSSG and abolished by deglutathionylation. The K(m) and k(cat) values for mitochondrial and cytosolic thioredoxins (9.5 microm and 131 s(-1), 34 microm and 197 s(-1), respectively) were higher than those reported for mammalian TRs. Analysis of TGR mutants revealed that the glutaredoxin domain is required for the GR activity but did not affect the TR activity. In contrast, both GR and TR activities were dependent on the Sec-containing redox center. The activity loss caused by the Sec-to-Cys mutation could be partially compensated by a Cys-to-Sec mutation of the neighboring residue, indicating that Sec can support catalysis at this alternative position. Consistent with the essential role of TGR in redox control, 2.5 microm auranofin, a known TGR inhibitor, killed larval worms in vitro. These studies establish the selenium- and glutathione-dependent regulation of cytosolic and mitochondrial redox homeostasis through a single TGR enzyme in platyhelminths.

SUBMITTER: Bonilla M

PROVIDER: S-EPMC2440607 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione.

Bonilla Mariana M Denicola Ana A Novoselov Sergey V SV Turanov Anton A AA Protasio Anna A Izmendi Darwin D Gladyshev Vadim N VN Salinas Gustavo G

The Journal of biological chemistry 20080411 26

Platyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate ...[more]

PMID: 18408002

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Project description:Most high M(r) thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys(1)-Cys(2)) which forms an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys(2) position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the eight-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from Drosophila melanogaster and Caenorhabditis elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group pK(a) as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower pK(a) of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to function at acidic pH.

Project description:Glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants in Sinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways in S. meliloti The first pathway is the de novo synthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether the S. meliloti GR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of the S. melilotigor gene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. The gor mutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H2O2 and sodium nitroprusside. Interestingly, the expression of gshA, gshB1, katA, and katB was induced in the mutant. These findings reveal that the recycling of glutathione is important for S. meliloti to maintain redox homeostasis and to interact symbiotically with alfalfa.IMPORTANCE The antioxidant glutathione is regulated by its synthetase and reductase in cells. In the symbiotic bacterium S. meliloti, the de novo synthesis of glutathione is essential for alfalfa nodulation and nitrogen fixation. In this study, we observed that the recycling of glutathione from GSSG not only was required for redox homeostasis and oxidative stress protection in S. meliloti cells but also contributed to alfalfa nodule development and competition capacity. Our findings demonstrate that the recycling of glutathione plays a key role in nitrogen fixation symbiosis.

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Dataset Information

Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione.

Publications

Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione.

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