Identification and Characterization ofgshA, a Gene Encoding the Glutamate-Cysteine Ligase in the Halophilic ArchaeonHaloferax volcanii

Abstract

Halophilic archaea were found to contain in their cytoplasm millimolar concentrations of ␥-glutamylcysteine (␥GC) instead of glutathione. Previous analysis of the genome sequence of the archaeon Halobacterium sp. strain NRC-1 has indicated the presence of a sequence homologous to sequences known to encode the glutamate-cysteine ligase GshA. We report here the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii and show that H. volcanii gshA directs in vivo the synthesis and accumulation of ␥GC. We also show that the H. volcanii gene when expressed in an Escherichia coli strain lacking functional GshA is able to restore synthesis of glutathione.Many organisms contain millimolar concentrations of lowmolecular-weight thiol compounds that participate in a number of important biological functions involving thiol-disulfide exchanges (7). In particular, they serve to maintain an intracellular reducing environment, to provide reducing power for key reductive enzymes, to combat the effects of oxidative and disulfide stress, and to detoxify xenobiotic compounds (7). Glutathione (GSH), a cysteine-containing tripeptide, L-␥-glutamyl-L-cysteinylglycine, is the best-characterized low-molecular-weight thiol (7,19,21). GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (3, 21). The ␥-carboxyl group of Lglutamate and the amino group of L-cysteine are ligated by the enzyme glutamylcysteine (GC) ligase EC 6.3.2.2 (GshA, encoded by gshA), which is then condensed with glycine in a reaction catalyzed by GSH synthetase (GshB, encoded by gshB) to form GSH (10,38). GSH is found primarily in gramnegative bacteria and eukaryotes and only rarely in grampositive bacteria (26). Fahey and coworkers showed that GSH is absent from the high-GC gram-positive actinomycetes which produce, as the major low-molecular-weight thiol, mycothiol, (13,(26)(27)(28)35). GSH is also absent in Archaea. In Pyrococcus furiosus, coenzyme A SH (CoASH) is the main thiol (11), whereas in Halobacterium salinarum, ␥GC is the predominant thiol and the organism possesses bis-␥GC reductase activity (30,36). Similarly, Leuconostoc kimchi and Leuconostoc mesenteroides, gram-positive lactic acid bacterial species, were recently found to contain ␥GC rather than GSH (15). To date, these are the sole procaryotic species reported to naturally produce ␥GC but not GSH (6,30). In this report, we describe the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii. Copley and Dhillon (6) previously identified, using bioinformatic tools, an open reading frame (ORF) (gene VNG1397C) in Halobacterium sp. strain NRC-1 with limited sequence relatedness to known GshA proteins (6). However, no genetic or biochemical evidence was presented to substantiate their conclusion. Here, we show that Haloferax volcanii strain DS2 (1, 25) contains an ORF that directs in vivo the synthesis and accumulation of ␥GC. We also show that the H. volcanii ORF, when expressed in Esche...

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