Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1.
ABSTRACT African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.
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ABSTRACT: Significance : Oxidative stress is widely invoked in inflammation, aging and complex diseases. To avoid unwanted oxidations, the redox environment of cellular compartments needs to be tightly controlled. The complementary action of oxidoreductases and of high concentrations of low molecular weight (LMW) non-protein thiols play an essential role in maintaining the redox potential of the cell in balance. Recent Advances : Whilst LMW thiols are central players in an extensive range of redox regulation/metabolism processes not all organisms use the same thiol cofactor(s) to this effect, as evidenced by the recent discovery of mycothiol and bacillithiol amongst different Gram-positive bacteria. Critical Issues : LMW thiol-disulfide exchange processes and their cellular implications are often oversimplified as only the biology of the free thiols and their symmetrical disulfides are considered. In bacteria under oxidative stress, especially where concentrations of different LMW thiols are comparable (eg, bacillithiol (BSH), Coenzyme A (CoA), and cysteine (Cys) in many low G+C Gram positive bacteria (Firmicutes)), mixed disulfides (eg. CoASSB, CySSCoA, etc) must surely be major thiol redox metabolites that need to be taken into consideration. Future Directions : There are many microorganisms whose low molecular weight thiol redox buffer(s) have not yet been identified (either bioinformatically or experimentally). Many elements of BSH and mycothiol (MSH) redox biochemistry remain to be explored. The fundamental biophysical properties, thiol pKa and redox potential, have not yet been determined, and the protein interactome in which the biothiols MSH and BSH are involved needs further exploration.Antioxidants & Redox Signaling 10/2012; · 8.20 Impact Factor
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ABSTRACT: Monothiol glutaredoxins (Grxs) play important roles in maintaining redox homeostasis in living cells and are conserved across species. Arabidopsis thaliana monothiol glutaredoxin AtGRXcp is critical for protection from oxidative stress in chloroplasts. The crystal structure of AtGRXcp has been determined at 2.4 A resolution. AtGRXcp has a glutaredoxin/thioredoxin-like fold with distinct structural features that differ from those of dithiol Grxs. The structure reveals that the putative active-site motif CGFS is well defined and is located on the molecular surface and that a long groove extends to both sides of the catalytic Cys97. Structural comparison and molecular modeling suggest that glutathione can bind in this groove and form extensive interactions with conserved charged residues including Lys89, Arg126 and Asp152. Further comparative studies reveal that a unique loop with five additional residues adjacent to the active-site motif may be a key structural feature of monothiol Grxs and may influence their function. This study provides the first structural information on plant CGFS-type monothiol Grxs, allowing a better understanding of the redox-regulation mechanism mediated by these plant Grxs.Acta Crystallographica Section D Biological Crystallography 06/2010; 66(Pt 6):725-32. · 12.67 Impact Factor
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ABSTRACT: Trypanosomes and leishmania, the causative agents of several tropical diseases, possess a unique redox metabolism which is based on trypanothione. The bis(glutathionyl)spermidine is the central thiol that delivers electrons for the synthesis of DNA precursors, the detoxification of hydroperoxides and other trypanothione-dependent pathways. Many of the reactions are mediated by tryparedoxin, a distant member of the thioredoxin protein family. Trypanothione is kept reduced by the parasite-specific flavoenzyme trypanothione reductase. Since glutathione reductases and thioredoxin reductases are missing, the reaction catalyzed by trypanothione reductase represents the only connection between the NADPH- and the thiol-based redox metabolisms. Thus, cellular thiol redox homeostasis is maintained by the biosynthesis and reduction of trypanothione. Nearly all proteins of the parasite-specific trypanothione metabolism have proved to be essential.Biochimica et Biophysica Acta 04/2008; 1780(11):1236-48. · 4.66 Impact Factor