Escherichia coli γ-Glutamylcysteine Synthetase

Kelly, Brenda S.; Antholine, William E.; Griffith, Owen W.

doi:10.1074/jbc.m107961200

Cited by 56 publications

(35 citation statements)

References 52 publications

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“…Next, the kinetic parameters were determined for both individual activities (summarized in Table 2). For comparison, Table 2 also includes the corresponding values for the S. agalactiae GshF (24) together with those of the E. coli monofunctional counterparts (4,42). It is interesting to note that no deviations from linearity were apparent in Eadie-Hofstee plots of the initial velocity data, indicating that both peptide bond-forming reactions are catalyzed by the bifunctional GshF without the involvement of cooperative mechanisms.…”

Section: Expression and Purification Of P Multocida Gshf In Ementioning

confidence: 99%

“…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 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. where Me 2ϩ again can be magnesium or manganese.…”

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confidence: 99%

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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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Section: Expression and Purification Of P Multocida Gshf In Ementioning

confidence: 99%

mentioning

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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“…On the other hand, Hiratake et al 29) suggested the importance of the length of the methylene chain of the substrate amino acid, which has a large influence on the hydrophobic interactions between the substrate and the enzyme. Kelly et al 25) reported that GCS from E. coli took 17 amino acids instead of Cys, and showed that the relative activity (V max =K m values) for amino acids was -aminobutylate (carbon chain length, 3) > alanine (2) > glycine (1) > norvaline (4). Based on our experiments, the relative activities of the amines depended on the length of the carbon chain as follows (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Recombinant E. coli DH5 was cultivated in LB medium (10 g/l Bacto-tryptone, BD (New Jersey, USA), 5 g/l yeast extract, BD, and 5 g/l NaCl) containing 100 mg/l of ampicillin at 30 C overnight with shaking. The culture was transferred to fresh LB medium and incubated at 25 C. Four h after transfer, 0.2 mM IPTG (isopropyl--D-thiogalactopyranoside) was added, and cultivation was continued for 16 h. Cells were collected by centrifugation, and the cell pellet was suspended in 50 mM phosphate buffer (pH 7.8) and disrupted by sonication at 4 C. The cellular debris was removed by centrifugation and the supernatant was subjected to further purification using HiTrapÔHP (GE Healthcare, Sweden). The protein concentration was determined using the Quick StartÔ Bradford Dye Reagent (Bio-Rad, California, USA) with bovine serum albumin as the standard.…”

Section: Methodsmentioning

confidence: 99%

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A Novel Catalytic Ability of γ-Glutamylcysteine Synthetase ofEscherichia coliand Its Application in Theanine Production

Miyake

Kakita

2009

Bioscience, Biotechnology, and Biochemistry

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-Glutamylcysteine synthetase (GCS, EC 6.3.2.2) catalyzes the formation of -glutamylcysteine from L-glutamic acid (Glu) and L-cysteine (Cys) in an ATPdependent manner. While GCS can use various amino acids as substrate, little is known about whether it can use non-amino acid compounds in place of Cys. We determined that GCS from Escherichia coli has the ability to combine Glu and amines to form -glutamylamides. The reaction rate depended on the length of the methylene chain of the amines in the following order: n-propylamine > butylamine > ethylamine methylamine. The optimal pH for the reaction was narrower and more alkaline than for the reaction with an amino acid. The newly found catalytic ability of GCS was used in the production of theanine (-glutamylethylamine). The resting cells of E. coli expressing GCS, in which ATP was regenerated through glycolysis, synthesized 12.1 mM theanine (18 h) from 429 mM ethylamine.

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Systematic engineering of phytochelatin synthesis and arsenic transport for enhanced arsenic accumulation in E. coli

Singh

Kang

Lee

et al. 2009

Biotech & Bioengineering

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Phytochelatin (PC) is a naturally occurring peptide with high affinity towards arsenic (As). In this article, we demonstrated the systematic engineering of PC-producing E. coli for As accumulation by addressing different bottlenecks in PC synthesis as well as As transport. Phytochelatin synthase from Schizosaccharomyces pombe (SpPCS) was expressed in E. coli resulting in 18 times higher As accumulation. PC production was further increased by co-expressing a feedback desensitized gamma-glutamylcysteine synthetase (GshI*), resulting in 30-fold higher PC levels and additional 2-fold higher As accumulation. The significantly increased PC levels were exploited further by co-expressing an arsenic transporter GlpF, leading to an additional 1.5-fold higher As accumulation. These engineering steps were finally combined in an arsenic efflux deletion E. coli strain to achieve an arsenic accumulation level of 16.8 micromol/g DCW, a 80-fold improvement when compared to a control strain not producing phytochelatins.

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Escherichia coli γ-Glutamylcysteine Synthetase

Cited by 56 publications

References 52 publications

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

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

A Novel Catalytic Ability of γ-Glutamylcysteine Synthetase ofEscherichia coliand Its Application in Theanine Production

Systematic engineering of phytochelatin synthesis and arsenic transport for enhanced arsenic accumulation in E. coli

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