A Multidomain Fusion Protein inListeria monocytogenesCatalyzes the Two Primary Activities for Glutathione Biosynthesis

Abstract

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, ␥-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate ␥-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a ␥-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.Glutathione (␥-glutamyl-cysteinyl-glycine) (GSH) is the predominant low-molecular-weight peptide thiol present in living organisms. In bacteria it plays a pivotal role in many metabolic processes, chief among which are thiol redox homeostasis, protection against reactive oxygen species, protein folding, and provision of electrons via NADPH to reductive enzymes, such as ribonucleotide reductase. Low-molecular-weight nonribosomal peptides are assembled by the action of versatile multimodular enzymes termed nonribosomal peptide synthetases (NRPS) (22, 37) or through the consecutive actions of individual enzymes. GSH synthesis is a prime example of the latter, and GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (21). The ␥-carboxyl group of L-glutamate and the amino group of L-cysteine are ligated by the enzyme ␥-glutamylcysteine ligase (encoded by gshA) to give ␥-glutamylcysteine, which is then condensed with glycine in a reaction catalyzed by glutathione synthetase (encoded by gshB) to form GSH. Most grampositive bacteria do not contain GSH (9). However, a broad survey of the distribution of thiols in microorganisms revealed that several species of gram-positive bacteria, including Listeria, streptococci, and enterococci, produce significant amounts of GSH (23). The source of GSH in these bacteria has remained a puzzle, since their genomes do not contain a canonical gshB gene. The recent paper of Copley and Dhillon provides a clue to the origin of this GSH (7). These authors identified in the genomes of Listeria monocytogenes, Listeria innocua, Clostridium perfringens, and Pasteurella multocida an open reading frame (ORF) that is predicted to co...

Characterization of the Bifunctional γ-Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida

“…A BLASTP search with 303 microbial genomes at NCBI (37) identified 19 species, of both Gram-negative and Gram-positive signature, containing a GshF homologous sequence. Among Gram-negative bacteria, the distribution of GshF sequences appears to be confined to (24,25) and, in case of L. monocytogenes, GshF was found to be essential for aerobic growth and virulence. Interestingly, some of the Gram-positive genera that harbor GshF, such as streptococci and enterococci, have been found to accumulate glutathione by either or both of the two presently recognized ways of glutathione acquisition, import and complete de novo synthesis (1,24,49).…”

“…The ATP-grasp fold residues of the aligned sequences shown in Fig. 3 exhibit overall similarity, although two comparable and major insertions are apparent in the C-terminal domains of the aligned CphA and GshF sequences compared with the DdlB sequences; these insertions cluster the former two proteins into a phylogenetically distinct branch of ATP-grasp fold-containing proteins (25). Nonetheless, the three loops that close over the catalytic cavity in the liganded E. coli DdlB structure are among the most conserved regions in the alignment, and, moreover, 8 of 10 DdlB MgATP 2Ϫ -binding residues are physicochemically conserved in both the CphA and GshF aligned sequences.…”

“…Moreover, conserved residues known to be involved in MgATP 2Ϫ binding in structurally characterized ATP-grasp enzymes are indicated in white on a purple background, of which 8 of 10 are also conserved in the GshF sequences. Following the ⍀-loop, GshF as well as CphA sequences exhibit similarly large insertions, which clusters them into a phylogenetically distinct branch of ATP-grasp proteins (25). thione (Fig.…”

“…proteins, found mostly in low GC Gram-positive bacteria, which catalyze the ATP-dependent formation of glutathione from the constituent amino acids L-Glu, L-Cys, and Gly, as demonstrated recently for the ␥-ECL⅐ATP-grasp fusions from Streptococcus agalactiae (24) and L. monocytogenes (25). As proposed for the L. monocytogenes ␥-ECL⅐ATP-grasp fusion (25), we refer to the P. multocida ␥-ECL⅐ATP-grasp fusion protein as GshF.…”

The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23K

“…These include two peptide methionine sulfoxide reductases (LSA0865, LSA0866), which counter methionine oxidation, and a redundancy of thioredoxin (LSA0218, LSA0307, LSA0394, LSA0634)/ thioredoxin reductase (LSA0403, LSA0435, LSA0520) systems. L. sakei possesses an additional glutathione/glutaredoxin (LSA0942)/ glutathione reductase (LSA1871) system, in which glutathione is presumably synthesized by the multidomain GshF protein 25 (LSA0840). Such a system seems to also be present in L. plantarum.…”