Three-dimensional Structure of the Glutathione Synthetase from Escherichia coli B at 2·0 Å Resolution

Yamaguchi, Hiroshi; Kato, Hiroaki; Hata, Yasuo; Nishi­oka, Takaaki; Kimura, Akira; Oda, Jun’ichi; Katsube, Yukiteru

doi:10.1006/jmbi.1993.1106

Cited by 125 publications

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“…Like other eukaryotic GSH synthetases, the yeast amino acid sequence shows limited homology to that of E. coli. The yeast protein shares 44% similarity and 18% identity with the bacterial enzyme but this does not include the proposed ATP or y-glutamylcysteine binding sites (Yamaguchi et al, 1993;Fan et al, 1995;Hara et al, 1995). The yeast sequence contains the highly conserved glycine-rich domains (Figure 1) that have been suggested to play an essential role in the catalytic activity of GSH synthetases (Ullmann et al, 1996).…”

Section: Determination Of Gsh Levels and Gshl And Gsh2 Enzyme Activitiesmentioning

confidence: 99%

Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

Grant

MacIver

Dawes

1997

MBoC

178

147

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Glutathione (GSH) synthetase (Gsh2) catalyzes the ATP-dependent synthesis of GSH from y-glutamylcysteine (y-Glu-Cys) and glycine. GSH2, encoding the Saccharomyces cerevisiae enzyme, was isolated and used to construct strains that either lack or overproduce Gsh2. The identity of GSH2 was confirmed by the following criteria: 1) the predicted Gsh2 protein shared 37-39% identity and 58-60% similarity with GSH synthetases from other eukaryotes, 2) increased gene dosage of GSH2 resulted in elevated Gsh2 enzyme activity, 3) a strain deleted for GSH2 was dependent on exogenous GSH for wild-type growth rates, and 4) the gsh2 mutant lacked GSH and accumulated the dipeptide y-Glu-Cys intermediate in GSH biosynthesis. Overexpression of GSH2 had no effect on cellular GSH levels, whereas overexpression of GSH1, encoding the enzyme for the first step in GSH biosynthesis, lead to an approximately twofold increase in GSH levels, consistent with Gshl catalyzing the rate-limiting step in GSH biosynthesis. In contrast to a strain deleted for GSH1, which lacks both GSH and y-Glu-Cys, the strain deleted for GSH2 was found to be unaffected in mitochondrial function as well as resistance to oxidative stress induced by hydrogen peroxide, tert-butyl hydroperoxide, and the superoxide anion. Furthermore, y-Glu-Cys was at least as good as GSH in protecting yeast cells against an oxidant challenge, providing the first evidence that -y-Glu-Cys can act as an antioxidant and substitute for GSH in a eukaryotic cell. However, the dipeptide could not fully substitute for the essential function of GSH in the cell as shown by the poor growth of the gsh2 mutant on minimal medium. We suggest that this function may be the detoxification of harmful intermediates that are generated during normal cellular metabolism.

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Section: Determination Of Gsh Levels and Gshl And Gsh2 Enzyme Activitiesmentioning

confidence: 99%

Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

Grant

MacIver

Dawes

1997

MBoC

178

147

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“…Crystal structures of the E. coli (13,14), human (15), and yeast (10) GS ligases, however, adopt a similar fold, which extends across two domains, collectively referred to as the ATP-grasp fold (16). Other members of the ATP-grasp enzyme superfamily include, among others, biotin carboxylase ␣-chain (17), succinate-CoA ligase (18), carbamoylphosphate synthetase (19), cyanophycin synthetase (CphA (20)), and D-Ala-D-Ala ligase (DdlB (21)), from which it can be appreciated that ATP-grasp enzymes carry out ATP-dependent carboxylate to amine/thiol ligase reactions in a number of unrelated biosynthetic pathways, accepting a wide variety of donor and acceptor substrates.…”

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confidence: 99%

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

Vergauwen

Vos

Beeumen

2006

Journal of Biological Chemistry

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Glutamate-cysteine ligase (␥-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. ␥-ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxylto-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steadystate levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal ␥-proteobacterial ␥-ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an ϳ85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The ␥-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (K i ‫؍‬ 13.6 mM). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.Glutathione (GSH; L-␥-glutamyl-L-cysteinylglycine) is the predominant low molecular weight peptide thiol present in many Gram-negative bacteria and in virtually all eukaryotes, except those that lack mitochondria (1, 2). Glutathione is made in a highly conserved two-step ATP-dependent biosynthesis pathway by two unrelated peptide bondforming enzymes (3). In the first and rate-limiting reaction, ␥-glutamate-cysteine ligase (␥-ECL) 2 (EC 6.3.2.2) condenses the ␥-carboxylate of L-glutamic acid with L-cysteine to form the dipeptide ␥-glutamylcys-where Me 2ϩ can be magnesium or manganese (4, 5). In the second step, ␥-EC is condensed with glycine in a reaction catalyzed by glutathione synthetase (GS; EC 6.3.2.3), according towhere Me 2ϩ again can be magnesium or manganese. The activity of eukaryotic ␥-ECL is precisely controlled by nonallosteric glutathione feedback inhibition, the limited availability of cellular L-Cys, and the transcriptional and posttranslational regulation of the expression and activity of the enzyme under various physiological conditions (6). Bacterial glutathione homeostasis is less well characterized, although glutathione feedback inhibition appears to be of major importa...

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“…One class of folds, the classic mononucleotide-binding fold (Schulz & Schirmer, 1974;Rossmann et al, 1975;Schulz et al, 1986), has an ATP-binding site placed at the edge of a parallel P-sheet. Two other classes of folds, the protein kinase family fold (Hanks et al, 1988;Hanks & Quinn, 1991;Hubbard et al, 1994;Xu et al, 1995), and the glutathione synthetase fold (Yamaguchi et al, 1993;Waldrop et al, 1994;Wolodko et al, 1994;Fan et al, 1995;Artymiuk et al, 1996;Herzberg et al, 1996;Hibi et al, 1996;Matsuda et al, 1996;Thoden et al, 1997;Esser et 1998), also called the "ATP-grasp'' fold (Murzin, 1996), have ATPbinding sites positioned on the surface of an antiparallel &sheet (Kobayashi & Go, 1997b). These last two fold classes are particularly interesting.…”

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Enzyme‐mononucleotide interactions: Three different folds share common structural elements for atp recognition

1998

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Three ATP-dependent enzymes with different folds, CAMP-dependent protein kinase, D-Ala:o-Ala ligase and the a-subunit of the a2P2 ribonucleotide reductase, have a similar organization of their ATP-binding sites. The most meaningful similarity was found over 23 structurally equivalent residues in each protein and includes three strands each from their @sheets, in addition to a connecting loop. The equivalent secondary structure elements in each of these enzymes donate four amino acids forming key hydrogen bonds responsible for the common orientation of the "AMP" moieties of their ATP-ligands. One lysine residue conserved throughout the three families binds the alpha-phosphate in each protein. The common fragments of structure also position some, but not all, of the equivalent residues involved in hydrophobic contacts with the adenine ring. These examples of convergent evolution reinforce the view that different proteins can fold in different ways to produce similar structures locally, and nature can take advantage of these features when structure and function demand it, as shown here for the common mode of ATP-binding by three unrelated proteins.Keywords: allosteric effector-binding site; ATP-dependent enzymes; convergent structural similarities; local structural comparisons; similar ligand-binding sites Adenosine 5"triphosphate (ATP) is necessary for the function of a wide variety of enzymes. Currently, the Brookhaven Protein Data Bank (PDB) (Bernstein et al., 1977) contains atomic coordinates for over 140 X-ray and NMR structures of ATP-dependent proteins solved as complexes mainly with bound ATP-analogues, bound AMP, bound ADP, and in some cases with bound ATP.Several distinct classes of folds of ATP-dependent enzymes have been identified (Schulz, 1992). Acharacteristic feature of these folds is the presence of a P-sheet, parallel or antiparallel, in the vicinity of the ATP-binding site. One class of folds, the classic mononucleotide-binding fold (Schulz & Schirmer, 1974;Rossmann et al., 1975;Schulz et al., 1986), has an ATP-binding site placed at the edge of a parallel P-sheet. Two other classes of folds, the protein kinase family fold (Hanks et al

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Three-dimensional Structure of the Glutathione Synthetase from Escherichia coli B at 2·0 Å Resolution

Cited by 125 publications

References 0 publications

Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

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

Enzyme‐mononucleotide interactions: Three different folds share common structural elements for atp recognition

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