Stress response of a clinical Enterococcus faecalis isolate subjected to a novel antimicrobial surface coating.

Emerging antibiotic resistance among pathogenic bacteria, paired with their ability to form biofilms on medical and technical devices, represents a serious problem for effective and long-term decontamination in health-care environments and gives rise to an urgent need for new antimicrobial materials. Here we present the impact of AGXX®, a novel broad-spectrum antimicrobial surface coating consisting of micro-galvanic elements formed by silver and ruthenium, on the transcriptome of Enterococcus faecalis. A clinical E. faecalis isolate was subjected to metal stress by growing it for different periods in presence of the antimicrobial coating or silver-coated steel meshes. Subsequently, total RNA was isolated and next-generation RNA sequencing was performed to analyze variations in gene expression in presence of the antimicrobial materials with focus on known stress genes. Exposure to the antimicrobial coating had a large impact on the transcriptome of E. faecalis. After 24min almost 1/5 of the E. faecalis genome displayed differential expression. At each time-point the cop operon was strongly up-regulated, providing indirect evidence for the presence of free Ag+-ions. Moreover, exposure to the antimicrobial coating induced a broad general stress response in E. faecalis. Genes coding for the chaperones GroEL and GroES and the Clp proteases, ClpE and ClpB, were among the top up-regulated heat shock genes. Differential expression of thioredoxin, superoxide dismutase and glutathione synthetase genes indicates a high level of oxidative stress. We postulate a mechanism of action where the combination of Ag+-ions and reactive oxygen species generated by AGXX® results in a synergistic antimicrobial effect, superior to that of conventional silver coatings.

Endophytic bacterium Sphingomonas SaMR12 promotes cadmium accumulation by increasing glutathione biosynthesis in Sedum alfredii Hance.

A hydroponic experiment was conducted to verify the effects of inoculation with endophytic bacteria Sphingomonas SaMR12 on root growth, cadmium (Cd) uptake, reactive oxygen species (ROS), antioxidases, glutathione (GSH) and the related gene expression of Sedum alfredii Hance under different levels of Cd such as 0, 10, 25, 100 and 400 µM. The results showed that inoculation of SaMR12 improved Cd accumulation and upregulated glutathione synthase (GS) expression, but slightly reduced malondialdehyde (MDA) concentration and alleviated Cd-induced damage in roots. However it didn't alter the activities of antioxidant enzymes. When Cd concentration exceeded 25 µM, SaMR12 increased the concentration of GSH and the expression level of GSH1. At high Cd treatment levels (100 and 400 µM), SaMR12 significantly reduced H2O2 concentration and enhanced expression level of 1-Cys peroxiredoxin PER1 and ATPS genes. These results indicate that although SaMR12 has no significant effects on antioxidases activities, it reduces H2O2 concentration by enhancing GSH concentration and relevant genes expression, and subsequently improves Cd tolerance and accumulation.

X-box binding protein 1 is essential for the anti-oxidant defense and cell survival in the retinal pigment epithelium.

Damage to the retinal pigment epithelium (RPE) is an early event in the pathogenesis of age-related macular degeneration (AMD). X-box binding protein 1 (XBP1) is a key transcription factor that regulates endoplasmic reticulum (ER) homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.

Nox4 regulates Nrf2 and glutathione redox in cardiomyocytes in vivo.

NADPH oxidase-4 (Nox4) is an important modulator of redox signaling that is inducible at the level of transcriptional expression in multiple cell types. By contrast to other Nox enzymes, Nox4 is continuously active without requiring stimulation. We reported recently that expression of Nox4 is induced in the adult heart as an adaptive stress response to pathophysiological insult. To elucidate the potential downstream target(s) regulated by Nox4, we performed a microarray screen to assess the transcriptomes of transgenic (tg) mouse hearts in which Nox4 was overexpressed. The screen revealed a significant increase in the expression of many antioxidant and detoxifying genes regulated by Nrf2 in tg compared to wild-type (wt) mouse hearts, and this finding was subsequently confirmed by Q-PCR. Expression of glutathione biosynthetic and recycling enzymes was increased in tg hearts and associated with higher levels of both GSH and the ratio of reduced:oxidised GSH, compared to wt hearts. The increases in expression of the antioxidant genes and the changes in glutathione redox effected by Nox4 were ablated in an Nrf2-null genetic background. These data therefore demonstrate that Nox4 can activate the Nrf2-regulated pathway, and suggest a potential role for Nox4 in the regulation of GSH redox in cardiomyocytes.

Hypoxia and kinase activity regulate lung epithelial cell glutathione.

The authors investigated the mechanisms by which hypoxia regulates glutathione (GSH) in lung epithelial cells, and specifically whether the mitogen-activated protein kinase (MAPK) system is involved in the response to hypoxia. Hypoxia decreased cellular GSH content and appeared to decrease the effect of N-acetylcysteine on repletion of GSH after hypoxia. Hypoxia decreased 2 key enzyme activities that regulate GSH synthesis, glutamate cysteine ligase (GCL) (E.C. 6.3.2.2) and glutathione synthase (GS) (E.C. 6.3.2.3). No hypoxia-dependent change occurred in GCL or GS protein expression on Western blots. When epithelial cells were transfected with an adenoviral vector that caused over expression of human catalase protein (Ad.Cat or Ad.mCat), GCL and GS activities did not decrease in hypoxia. Inhibition of p38(MAPK) (using SB203580) or extracellular signal-regulated kinase (ERK; PD98059) prevented the hypoxia-dependent decrease in GCL and GS activity. To seek in vivo correlation, the authors assayed total glutathione in lungs and livers from MK2(-/-) (homozygous knockout) mice. MK2(-/-) mice are presumably unable to phosphorylate heat shock protein 27 (Hsp27) normally, because of absent kinase (MK2) activity. Liver GSH content (expressed per mg protein) was 20% less in MK2(-/-) mice than in nontransgenic Black 6 controls. Down-regulation of lung GSH content in hypoxia depends on peroxide tone of the cell and the p38(MAPK) system.

Expression of glutathione S-transferase pi and glutathione synthase correlates with survival in early stage non-small cell carcinomas of the lung.

The glutathione S-transferase (GST) family of genes encode for detoxification enzymes that protect against reactive oxygen species and influence host susceptibility to carcinogens, including tobacco smoke. It has not been determined whether isoenzyme GST-pi or glutathione synthase (GSH2) expression by tumor cells bears a relationship to survival. A total of 201 non-small cell lung cancers (NSCLC) with long-term follow-up were immunostained with antibodies to GST-pi and GSH2 using standard immunostaining techniques. Results were graded semiquantitatively using a scale of 0 to 3 (0 < or = 10%; 1 = 10%-50%; 2 = 51%-80%; 3 > or = 80%) for both nuclear and cytoplasmic staining. Results were correlated with patient survival using Kaplan-Meier analysis. Nuclear staining with GST-pi in greater than 10% of the cells was closely associated with decreased survival (P = .02) in stage I and II squamous cell carcinomas (n = 40). Cytoplasmic staining showed a similar trend that did not reach statistical significance. No significant correlation between GST-pi staining and survival was determined for other histologic types of NSCLC. Cytoplasmic GSH2 staining in greater than 80% of tumor cells was associated with a trend toward improved survival for stage I adenocarcinoma (P = .08) but did not show a relationship to survival for other histologic types of NSCLC. GST-pi expression predicts prognosis in stage I and II squamous cell lung carcinoma, and GSH2 expression may indicate better survival in early stage adenocarcinoma of the lung. Manipulation of GST-pi and GSH2 may be a potential basis for treatment of some NSCLC.

Role of protein kinase C delta in curcumin-induced antioxidant response element-mediated gene expression in human monocytes.

The Nrf2/antioxidant response element (ARE) signaling pathway plays a key role in activating cellular antioxidants, including heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase-1 (NQO1), and glutathione. Protein kinase C (PKC) may also regulate these antioxidants, as PKC phosphorylates Nrf2 in vitro. This study examined the role of PKC in ARE-mediated gene regulation in human monocytes by curcumin, a potent inducer of the Nrf2/ARE pathway. Curcumin increased HO-1 and glutamyl cysteine ligase modulator (GCLM) expression and stimulated Nrf2 binding to the ARE. Curcumin also rapidly stimulated PKC phosphorylation and Ro-31-8220, a pan-PKC inhibitor, decreased curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Rottlerin (a PKC delta inhibitor) and PKC delta antisense oligonucleotides significantly inhibited curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Furthermore, a p38 MAP kinase inhibitor reduced GCLM and HO-1 expression and rottlerin inhibited curcumin-induced p38 phosphorylation. In summary, curcumin activates ARE-mediated gene expression in human monocytes via PKC delta, upstream of p38 and Nrf2.

Molecular changes in Pisum sativum L. roots during arbuscular mycorrhiza buffering of cadmium stress.

Molecular responses to cadmium (Cd) stress were studied in mycorrhizal and non-mycorrhizal Pisum sativum L. cv. Frisson inoculated with Glomus intraradices. Biomass decreases caused by the heavy metal were significantly less in mycorrhizal than in non-mycorrhizal plants. Real-time reverse transcriptase-polymerase chain reaction showed that genes implicated in pathways of Cd detoxification varied in response to mycorrhiza development or Cd application. Expression of a metallothionein-encoding gene increased strongly in roots of Cd-treated non-mycorrhizal plants. Genes encoding gamma-glutamylcysteine synthetase and glutathione (GSH) synthetase, responsible for the synthesis of the phytochelatin (PC) precursor GSH, were activated by Cd in mycorrhizal and non-mycorrhizal plants. Cd stress decreased accumulation of GSH/homoglutathione (hGSH) and increased thiol groups in pea roots, whether mycorrhizal or not, suggesting synthesis of PCs and/or homophytochelatins. An hGSH synthetase gene, involved in hGSH synthesis, did not respond to Cd alone but was activated by mycorrhizal development in the presence of Cd. Transcript levels of a glutathione reductase gene were only increased in non-mycorrhizal roots treated with Cd. Studies of three stress-related genes showed that a heat-shock protein gene was activated in mycorrhizal roots or by Cd and chitinase gene transcripts increased under Cd stress to a greater extent in mycorrhizal roots, whilst a chalcone isomerase gene was only up-regulated by Cd. Results indicate that although heavy metal chelation pathways contribute to Cd stress responses in pea, they may not make a major contribution to Cd tolerance strategies operating in the arbuscular mycorrhizal symbiosis.

Glutathione metabolism during aging and in Alzheimer disease.

The concentration of glutathione (GSH), the most abundant intracellular nonprotein thiol and important antioxidant, declines with age and in some age-related diseases. The underlying mechanism, however, is not clear. The previous studies from our laboratory showed that the age-dependent decline in GSH content in Fisher 344 rats was associated with a downregulation of glutamate cysteine ligase (GCL), the rate-limiting enzyme in de novo GSH synthesis. Our recent studies further indicated that the activity and mRNA content of glutathione synthase (GS), which catalyzes the second reaction in de novo GSH synthesis, were also decreased with age in some tissues. No age-associated change was observed in glutathione reductase or gamma-glutamyl transpeptidase activities. Also, although GSH content declined with age in both male and female mice, male mice experienced more dramatic age-associated decline in many tissues/organs than female mice. Furthermore, we found that GSH content was significantly decreased in the red blood cells from male Alzheimer disease patients, which was associated with decreases in GCL and GS activities. Finally, we showed that estrogen increased GSH content, GS and GR activities, and GCL gene expression in the liver of both male and female mice. Taken together, our results suggest that (1) GCL plays a critical role in maintaining GSH homeostasis under both physiological and pathological conditions; (2) decreased GSH content may be involved in AD pathology in humans; and (3) estrogen increases GSH content in mice by multiple mechanisms.

Increased transcription and activity of glutathione synthase in response to deficiencies in folate, vitamin E, and apolipoprotein E.

Oxidative stress is a major contributing factor in neurodegeneration and can arise from dietary, environmental, and genetic sources. Here we examine the separate and combined impact of deprivation of folate and vitamin E, coupled with dietary iron as a prooxidant, on normal mice and transgenic mice lacking apolipoprotein E (ApoE-/- mice). Both mouse strains exhibited increased levels of glutathione when deprived of folate and vitamin E, but a substantial further increase was observed in ApoE-/- mice. To determine the mechanism(s) underlying this increase, we quantified transcription and activity of glutathione synthase (GS). Both normal and ApoE-/- mice demonstrated increased GS activity when deprived of folate and vitamin E. However, transcription was increased only in ApoE-/- mice deprived of folate and vitamin E. These findings demonstrate that deficiency in one gene can result in compensatory up-regulation in a second relevant gene and, furthermore, indicate that compensation for oxidative stress can occur in brain tissue at epigenetic and genetic levels depending on the nature and/or extent of oxidative stress.

The glutathione synthetase of Schizosaccharomyces pombe is synthesized as a homodimer but retains full activity when present as a heterotetramer.

Glutathione synthetase was overexpressed as a histidine-tagged protein in Schizosaccharomyces pombe and purified by two-step affinity chromatography. The recovered enzyme occurred in two different forms: a homodimeric protein consisting of two identical 56-kDa subunits and a heterotetrameric protein composed of two 32-kDa and two 24-kDa subfragments. Both forms are encoded by the GSH2 gene. The 56-Da protein corresponds to the complete GSH2 open reading frame, while the subfragments are produced following the cleavage of this larger protein by a metalloprotease. A stable homodimer was obtained by site-directed mutagenesis to remove the protease cleavage site, and this showed normal activity. A structural model of the fission yeast glutathione synthetase was produced, based on the x-ray coordinates of the human enzyme. According to this model the interacting domains of the proteolytic subfragments are strongly entangled. The subfragments were therefore coexpressed as independent proteins. These subfragments assembled correctly to yield functional heterotetramers with equivalent activity to the wild type enzyme. Furthermore, a permuted version of the protein was created. This also showed normal levels of glutathione synthetase activity. These data provide novel insight into the mechanisms of protein folding and the structure and evolution of the glutathione synthetase family.

Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance.

Cisplatin is a potent cytotoxic agent that functions as a bivalent electrophile, forming both interstrand and intrastrand DNA cross-links. Cisplatin-mediated DNA damage results in cell cycle arrest and initiation of apoptotic cell death. Increased cellular glutathione concentrations have been closely correlated with cisplatin resistance but do not reduce the extent of cisplatin-DNA adduct formation. One hypothesis to explain the ability of glutathione to inhibit cisplatin cytotoxicity is that glutathione, through its antioxidant function, plays a role in apoptotic regulatory pathways. We tested this hypothesis using MCF-7 breast cancer cells transfected with the apoptotic inhibitor Bcl-2. Bcl-2 overexpression in MCF-7 cells was associated with a nearly 3-fold increase in cellular glutathione levels and with increased resistance to cell death after cisplatin exposure. Treatment of MCF-7 lines with buthionine sulfoximine, an inhibitor of glutathione synthesis, normalized glutathione levels in Bcl-2 and control transfectants and completely abrogated Bcl-2-mediated cisplatin resistance without affecting Bcl-2 expression. Bcl-2 overexpression and up-regulation of glutathione were not associated with a change in either cisplatin-DNA adduct formation or repair over time. These results suggest that Bcl-2-mediated cisplatin resistance in MCF-7 cells is dependent on up-regulation of glutathione production, which contributes to cell survival by mechanisms independent of cisplatin inactivation or inhibition of DNA adduct formation. A similar dependence on glutathione for Bcl-2-mediated inhibition of cisplatin toxicity was confirmed in a second cell line, the lymphocytic precursor FL5.12. Taken together, these data suggest that apoptotic signaling after genotoxic exposure can be inhibited by the antioxidant activity of glutathione. Inhibition of glutathione synthesis or modulation of glutathione stores in tumors that overexpress Bcl-2 may comprise a novel anticancer strategy.

Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium.

DNA microarray technology enables genome-wide detection of cell response at the transcriptional level. We are planning to make bioassay systems that can detect environmental chemicals to screen for potential bioreactive agents. To develop a DNA microarray for our purposes, the changes in gene expression underlying the yeast stress response to cadmium were analyzed by a microarray of total mRNA. Cadmium is a potent cell poison known to cause oxidative stress by changing intracellular glutathione levels. We report here that not only the glutathione synthesis gene (GSH1) but also almost all transcripts of the enzymes involved in the sulfur amino acid metabolism, especially MET14 and MET17, were greatly induced after exposure to cadmium. While several common stress-responsive genes, such as HSP26, GRE1, HSP12, and DDR48, were up-regulated more than almost fourfold by cadmium, there were also 42 other genes up-regulated more than fourfold. Based on these results, we concluded that DNA microarrays are very useful instruments for creating new bioassay systems and finding genetic promoters of stress indicators.

Short- and long-term effects of dehydroascorbate in Lupinus albus and Allium cepa roots.

Administration of 1 mM dehydroascorbate (DHA) results in a rapid and large increase in cellular ascorbate (AA) content in both Lupinus albus L. and Allium cepa L. root tips. Uptake of DHA from the medium occurs at a high rate within 10-12 h of incubation, and is slowed down thereafter. In the first few h, DHA reduction to AA is apparently correlated to GSH depletion and slightly higher DHA reductase activity. DHA incubation also seems to induce new GSH synthesis. Longer DHA incubation (24 h) affects root growth by inhibiting cell proliferation. At this stage, an apparently generalised oxidation of SH-containing proteins is observed in DHA-treated roots. Treatment with 1 mM L-galactono-gamma-lactone, the last precursor of AA biosynthesis, results in an increase in AA content similar to that obtained with DHA, but stimulates growth and affects the redox state of SH-containing proteins in the opposite way. A possible multi-step mechanism of DHA reduction/removal is suggested and the hypothesis that DHA inhibits cell cycle progression by affecting the redox state of SH-containing proteins is discussed.

[Cloning and expression of the genes of glutathione synthetases].

The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively. The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh. E. coli BL21 was transformed by pTrc-gsh for expression of the related enzymes. Analysis of SDS-PAGE showed that the expected products were expressed. E. coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5. The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG. The expressed products were up to 25% of the total protein of the bacteria. Acetone-treated cells of the engineered strain could synthesize GSH efficiently.

Inducers of gamma-glutamylcysteine synthetase and their effects on glutathione synthetase expression.

Synthesis of GSH occurs via two enzymatic steps, the first is catalyzed by gamma-glutamylcysteine synthetase (GCS) and the second is catalyzed by GSH synthetase (GS). A heavy (HS) and light subunit (LS) make up GCS; regulation of both subunits have been well characterized, whereas regulation of GS is largely unknown. In this study, we examined the effects of treatments known to influence the gene expression of GCS subunits on GS expression. Insulin and hydrocortisone treatment of rat hepatocytes or ethanol-feeding of rats for 9 weeks, which increased the expression of GCS-HS only, had no influence on GS expression. However, two-thirds partial hepatectomy in rats which increased the expression of GCS-HS only, also increased GS expression. Treatment of hepatocytes or rats with diethyl maleate, buthionine sulfoximine, tert-butylhydroquinone, or thioacetamide, which increased the expression of both GCS subunits, increased the expression of GS. The GSH synthesis capacity increased 50-100% by treatments that increased only the GCS-HS expression, whereas it increased 161-200% by treatments that increased both GCS-HS and GS expression. Thioacetamide treatment of Chang cells increased cell GSH and GS expression by 50%, but had minimal influence on GCS subunits. Thus, GS induction can further increase the cell's GSH synthetic capacity and in some cells may be as important as GCS in determining the rate of GSH synthesis.

Molecular identification of glutathione synthetase (GSH2) gene from Saccharomyces cerevisiae.

The hypothetical protein YOL049w on the chromosome XV was identified to be the structural gene for glutathione synthetase (GSH2) of Saccharomyces cerevisiae. Translational initiation site was identified by making the GSH2-lacZ fusion. The GSH2 gene contained an open reading frame (1473 bp) with 491 amino acids, and molecular weight of the GSH2 gene product was calculated to be 55,812. Glutathione synthetase activity in transformant carrying the GSH2 gene with multicopy plasmid increased approximately 4-fold. The GSH2 gene was not essential for growth of yeast cell, and glutathione was not detected from the gsh2 disrupter.

Induction of glutathione synthetase by 1,10-phenanthroline.

The differential display (DD) was employed to identify the gene(s) responsible for 1,10-phenanthroline (OP)-induced apoptosis in murine tumor cells (Sun, Y., Bian, J., Wang, Y. and Jacobs, C. (1997) Oncogene 14, 385-393 [1]). An OP-inducible gene was isolated which encodes mouse glutathione synthetase (GSS). The GSS mRNA level began to increase 6 h post OP treatment and remained at a high level thereafter up to 24 h tested. Induction of GSS was found not to be associated with p53 activation. No significant induction of DNA fragmentation was detected in two murine tumor lines upon GSS transfection. This is the first observation indicating that GSS is inducible rather specifically by a metal chelator and that induction of GSS, however, is not sufficient to induce apoptosis. It may merely reflect a cellular response to OP-induced redox disturbance.

Modification of thiol contents in poplars (Populus tremula x P. alba) overexpressing enzymes involved in glutathione synthesis.

The hybrid poplar (Populus tremula x P. alba) was transformed to express the Escherichia coli gene for gamma-glutamylcysteine synthetase (EC 6.3.2.2: gamma-ECS) in the cytosol. Four transformed lines of poplar were obtained. These were phenotypically indistinguishable from untransformed poplars. Three lines, ggs28 (Noctor et al. 1996, Plant Physiol 112: 1071-1078), ggs11 and ggs5 possessed high levels of bacterial gene transcripts. Line ggs17 had lower transcript levels. Antisera were prepared against bacterial gamma-ECS and bacterial glutathione synthetase (EC 6.3.2.3: GS). Using the antiserum prepared against the purified His-tagged E. coli gamma-ECS, lines ggs28, ggs11 and ggs5 were shown to possess abundant quantities of the bacterial protein, whereas ggs17 contained lower amounts. The antiserum prepared against the purified His-tagged E. coli GS was also effective in screening poplars transformed with the E. coli gene coding for this enzyme. Immunoblots of leaf extracts from poplars overexpressing GS using this antibody revealed two bands. The extractable foliar gamma-ECS activities of the gamma-ECS transformants were in quantitative agreement with the protein levels. Lines ggs28, ggs11 and ggs5 had approximately 30-fold higher gamma-ECS activity than untransformed poplars, whereas in ggs17 this activity was only augmented about 3-fold. The lines strongly overexpressing gamma-ECS, ggs28, ggs11 and ggs5, contained enhanced foliar levels of cysteine (up to 2-fold), gamma-glutamylcysteine (5- to 20-fold) and glutathione (2- to 4-fold). Foliar thiol contents in ggs17 were no different to those of untransformed plants.

Flexible loop that is novel catalytic machinery in a ligase. Atomic structure and function of the loopless glutathione synthetase.

The catalytic mechanism of glutathione synthetase is proposed to proceed via phosphorylation of the dipeptide substrate to yield an acyl phosphate intermediate; this intermediate is subsequently attacked by glycine, followed by loss of inorganic phosphate, leading to glutathione formation. A flexible loop (Ile226-Gly242) in Escherichia coli B glutathione synthetase is proposed to stabilize the acyl phosphate intermediate by preventing its decomposition by hydrolysis with water [Tanaka, T., Kato, H., Nishioka, T., & Oda, J. (1992) Biochemistry 31, 2259-2265; Tanaka, T., Yamaguchi, H., Kato, H., Nishioka, T., Katsube, Y., & Oda, J. (1993) Biochemistry 32, 12398-12404]. To investigate the function of the loop in the E. coli enzyme definitely, a loopless mutant in which the loop (Ile226-Arg241) was replaced with three residues of glycine was constructed. The crystal structure of the loopless mutant enzyme was essentially identical with that of the wild-type enzyme. Kinetic measurements showed that the replacement of the loop led to increases in the Km values, especially for the glycine, and a 930-fold decrease in the k0 value. Hence, the loopless mutant was 3 x 10(4) less active in terms of its specificity constant (k0/Km) for glycine than the wild-type enzyme. Moreover, the loopless mutant showed gamma-L-glutamyl-L-cysteine-dependent ATP hydrolase activity to almost the same extent as its glutathione synthetase activity. These studies support the fact that the loop enhances the recognition of glycine as well as stabilizes the acyl phosphate intermediate so that the intermediate rapidly reacts with glycine.