Abstract
Among the Chromatiaceae, the glutathione derivative gamma-l-glutamyl-l-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined. The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase. Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH. By establishing Michaelis constants of 97 and 13.2 microm for glutathione amide disulfide and NADH, respectively (in contrast to K(m) values of 6.9 mm for glutathione disulfide and 1.98 mm for NADPH), the exclusive substrate specificities of GAR have been documented. Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21. On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2'-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.
Highlights
Among the Chromatiaceae, the glutathione derivative ␥-L-glutamyl-L-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species
Computer alignment of the entire Brassica rapa CPrxII, a Prx peroxidase that has been shown to catalyze the reduction of hydrogen peroxide with the use of electrons from the thioredoxin system [33], with amino acid residues 1–163 of the garA deduced chimeric protein reveals 39% identity and striking homology around the Nterminal cysteine residue, which is believed to be the site of oxidation by peroxides [36]
The work reported here demonstrates that the activity is carried out by a new member of the class I flavoprotein disulfide reductase family of oxidoreductases, which includes GR [43, 44], trypanothione reductase [45], mycothione reductase [46], ␥-glutamylcysteine reductase [9], mercuric reductase [47, 48], and lipoamide dehydrogenase [49]
Summary
Among the Chromatiaceae, the glutathione derivative ␥-L-glutamyl-L-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. In eukaryotes, some reactive oxygen intermediates are detoxified directly by the action of glutathione peroxidases [6] and, to a lesser extent, of glutathione S-transferases [7] Members of another enzyme family of GSH-dependent thiol-disulfide oxidoreductases, designated thioltransferases or glutaredoxins (Grx), are believed to act as one of the primary defenders against mixed disulfides formed after oxidative damage to proteins [8]. Detoxification of the freely diffusible hydrogen peroxide (H2O2), which in turn can be reduced further via the Fenton reaction to extremely reactive hydroxyl radicals, is completed by the action of catalases, heme- and manganesecontaining peroxidases, and several members of the large multifunctional AhpC/TSA protein family, recently classified as peroxiredoxins (Prx) To date, it seems that all bacterial Prx. Chromatium gracile GAR and Novel Prx/Grx Hybrid Peroxidase enzymes obtain the necessary reducing equivalents from the thioredoxin reducing system itself or from a thioredoxin-like reducing system, because it was demonstrated recently that the flavoprotein component (AhpF) of the Salmonella typhimurium alkyl hydroperoxide reductase (AhpCF) system and bacterial thioredoxin reductase have very similar mechanistic properties [15]. Chromatium species extracts do show glutathione amide disulfide (GASSAG) reductase activity [16], and the involvement of GAR as the heterodisulfide reductase in the hypothesized sulfide transfer mechanism has to be considered because GSH persulfide reduction was established already for the bovine GR [18]
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