Synthesis of γ-glutamylcysteine as a major low-molecular-weight thiol in lactic acid bacteria Leuconostoc spp.

Kim, Eunkyoung; Cha, Chang-Jun; Cho, Yong‐Joon; Cho, Yoo-Bok; Roe, Jung‐Hye

doi:10.1016/j.bbrc.2008.02.139

Cited by 30 publications

(24 citation statements)

References 25 publications

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“…Therefore, the major intracellular thiol of L. reuteri is unlikely to be glutathione but may be ␥-glutamylcysteine, as has previously been shown to occur in Leuconostoc spp. (24). The question as to whether L. reuteri is capable of reducing oxidized ␥-glutamylcysteine remains open.…”

Section: Discussionmentioning

confidence: 99%

Cystathionine γ-Lyase Is a Component of Cystine-Mediated Oxidative Defense in Lactobacillus reuteri BR11

Turner

Barry

et al. 2009

J Bacteriol

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Lactobacillus reuteri BR11 possesses a novel mechanism of oxidative defense involving an abundant cystine ABC transporter encoded by the cyuABC gene cluster. Large amounts of thiols, including H 2 S, are secreted upon cystine uptake by the CyuC transporter. A cystathionine ␥-lyase (cgl) gene is cotranscribed with the cyu genes in several L. reuteri strains and was hypothesized to participate in cystine-mediated oxidative defense by producing reducing equivalents. This hypothesis was tested with L. reuteri BR11 by constructing a cgl mutant (PNG901) and comparing it to a similarly constructed cyuC mutant (PNG902). Although Cgl was required for H 2 S production from cystine, it was not crucial for oxidative defense in de Mann-Rogosa-Sharpe medium, in contrast to CyuC, whose inactivation resulted in lag-phase arrest in aerated cultures. The importance of Cgl in oxidative defense was seen only in the presence of hemin, which poses severe oxidative stress. The growth defects in aerated cultures of both mutants were alleviated by supplementation with cysteine (and cystine in the cgl mutant) but not methionine, with the cyuC mutant showing a much higher concentration requirement. We conclude that L. reuteri BR11 requires a high concentration of exogenous cysteine/cystine to grow optimally under aerobic conditions. This requirement is fulfilled by the abundant CyuC transporter, which has probably arisen due to the broad substrate specificity of Cgl, resulting in a futile pathway which degrades cystine taken up by the CyuC transporter to H 2 S. Cgl plays a secondary role in oxidative defense by its well-documented function of cysteine biosynthesis.

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Section: Discussionmentioning

confidence: 99%

Cystathionine γ-Lyase Is a Component of Cystine-Mediated Oxidative Defense in Lactobacillus reuteri BR11

Turner

Barry

et al. 2009

J Bacteriol

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“…In their studies of ␥GC formation in Halobacterium halobium, Sundquist and Fahey hypothesized that halophiles might utilize ␥GC rather than GSH since they found that ␥GC is at least as stable to metal-catalyzed oxidation in high salt concentrations as GSH, which is not the case in low salt concentrations (36). It is interesting to note therefore that nonhalophiles L. kimchi and L. mesenteroides make large amounts of ␥GC but lack GSH (15). Further studies are required to elucidate the particular advantage(s), if at all, related to why some bacteria employ GSH in place of the simpler compound ␥GC.…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

et al. 2009

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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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“…Although little is known about the physiological role of GSH in Gram-positive lactobacilli, some Lactobacillus species do possess gshA and gshAB (Kim et al 2008) suggesting that some may synthesize GSH or take it up from the medium. The resistance of L. lactis to various environmental stresses includes oxidative shock (Li et al 2003) and acid stress (Zhang et al 2007) but the GSH responses of Lactobacillus species to various environmental stresses have not been investigated.…”

Section: Introductionmentioning

confidence: 98%

Glutathione-mediated response to acid stress in the probiotic bacterium, Lactobacillus salivarius

Lee

Kim

et al. 2010

Biotechnol Lett

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Lactobacillus salivarius, a probiotic bacterium, encounters acidic conditions in its passage through the gastrointestinal tract of human and animal hosts. We studied the effect of a rapid downshift in extracellular pH from 6.5 to 4 on cell growth. The maximum growth rate was higher in low pH medium with glutathione supplementation than without. Cells developed a GSH-mediated acid-tolerance response and, when grown with 0.5 mM GSH, reached a higher final density than with other conditions. These findings suggest that the increased growth rate is caused by uptake of GSH which acts as a nutrient source as well as having protective functions, allowing for continued growth.

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Synthesis of γ-glutamylcysteine as a major low-molecular-weight thiol in lactic acid bacteria Leuconostoc spp.

Cited by 30 publications

References 25 publications

Cystathionine γ-Lyase Is a Component of Cystine-Mediated Oxidative Defense in Lactobacillus reuteri BR11

Cystathionine γ-Lyase Is a Component of Cystine-Mediated Oxidative Defense in Lactobacillus reuteri BR11

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

Glutathione-mediated response to acid stress in the probiotic bacterium, Lactobacillus salivarius

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