Glutathione is a physiologic reservoir of neuronal glutamate

Glutamate, the principal excitatory neurotransmitter of the brain, participates in a multitude of physiologic and pathologic processes, including learning and memory. Glutathione, a tripeptide composed of the amino acids glutamate, cysteine, and glycine, serves important cofactor roles in antioxidant defense and drug detoxification, but glutathione deficits occur in multiple neuropsychiatric disorders. Glutathione synthesis and metabolism are governed by a cycle of enzymes, the γ-glutamyl cycle, which can achieve intracellular glutathione concentrations of 1–10 mM. Because of the considerable quantity of brain glutathione and its rapid turnover, we hypothesized that glutathione may serve as a reservoir of neural glutamate. We quantified glutamate in HT22 hippocampal neurons, PC12 cells and primary cortical neurons after treatment with molecular inhibitors targeting three different enzymes of the glutathione metabolic cycle. Inhibiting 5-oxoprolinase and γ-glutamyl transferase, enzymes that liberate glutamate from glutathione, leads to decreases in glutamate. In contrast, inhibition of γ-glutamyl cysteine ligase, which uses glutamate to synthesize glutathione, results in substantial glutamate accumulation. Increased glutamate levels following inhibition of glutathione synthesis temporally precede later effects upon oxidative stress.     

Dipeptide precursor of garlic odour in Marasmius species

γ-Glutamyl-marasmine, a new natural dipeptide containing an unusual cysteine sulphoxide moiety has been isolated from the Basidiomyceteous mushrooms Marasmius alliaceus, M. scorodonius and M. prasiosmus, which are known for their garlic like odour. It is shown that this compound is the common natural precursor and that its two step enzymatic cleavage leads to the odorous substances. In the first step γ-glutamyl -marasmine is cleaved by a γ-glutamyl transpeptidase. The formed marasmine is split in a second enzymatic reaction by a C-S lyase into pyruvic acid, ammonia and an unstable sulfur compound, which decomposes to form the odorous secondary products.     

Transport of the large-neutral amino acids by the gamma-glutamyl cycle: a proposal.

The γ-glutamyl cycle has been proposed by Meister (1973) as one possible mechanism for the mediation of amino acid transport. The high energy requirement of the pathway, the very low specificity of γ-glutamyl transpeptidase and the inability to account for trans membrane stimulation of amino acid entry are but three criticisms of this hypothesis. It is proposed that the various objections can be overcome by postulating that the soluble form of γ-glutamyl transpeptidase transfers the γ-glutamyl moiety from gluthathione to glutamine (in the case of brain) and that the membrane sequestered form of this enzyme catalyzes the exchange of the γ-glutamyl group between γ-glutamyl glutamine and an entering neutral amino acid. The released glutamine leaves the cell. The γ-glutamyl amino acid then passes into the cytoplasm where it is acted upon by either γ-glutamyl cyclotransferase or the soluble γ-glutamyl transpeptidase which transfers the γ-glutamyl group to another molecule of glutamine. It is postulated that access to the membrane-bound enzyme is dependent on the relative lipophilia of the entering large-neutral amino acids. The available data support this mechanism. By regeneration of γ-glutamyl glutamine, a low expenditure of energy is required for the transport process. Specificity of transpeptidation is attained by the constraints of access to the membrane bound enzyme site.     

Pathways of glutathione degradation in the yeast Saccharomyces cerevisiae

The degradation of glutathione (GSH) in the yeast Saccharomyces cerevisiae appears to be mediated only by γ-glutamyltranspeptidase and cysteinylglycine dipeptidase. Other enzymes of the γ-glutamyl cycle, γ-glutamyl cyclotransferase and 5-oxo-l-prolinase, are not present in the yeast. In vivo transpeptidation was shown in the presence of a high intracellular level of γ-glutamyltranspeptidase, but only when the de-repressing nitrogen source was a suitable acceptor of the transferase reaction. In contrast, when the de-repressing source was not an acceptor of the transferase reaction (e.g. urea), only glutamate was detected. Intracellular GSH is virtually inert when the level of γ-glutamyltranspeptidase is low. Possible roles for in vivo transpeptidation are discussed.     

Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways

The first and highly conserved step in glutathione (GSH) biosynthesis is formation of γ-glutamyl cysteine by the enzyme glutamate-cysteine ligase (GshA). However, bioinformatic analysis revealed that many prokaryotic species that encode GSH-dependent proteins lack the gene for this enzyme. To understand how bacteria cope without gshA, we isolated Escherichia coli ΔgshA multigenic suppressors that accumulated physiological levels of GSH. Mutations in both proB and proA, the first two genes in L-proline biosynthesis, provided a new pathway for γ-glutamyl cysteine formation via the selective interception of ProB-bound γ-glutamyl phosphate by amino acid thiols, likely through an S-to-N acyl shift mechanism. Bioinformatic analysis suggested that the L-proline biosynthetic pathway may have a second role in γ-glutamyl cysteine formation in prokaryotes. Also, we showed that this mechanism could be exploited to generate cytoplasmic redox buffers bioorthogonal to GSH.     

γ-Glutamyl transpeptidase architecture: Effect of extra sequence deletion on autoprocessing,structure and stability of the protein from Bacillus licheniformis

γ-Glutamyl transpeptidases (γ-GTs, EC 2.3.2.2) are a class of ubiquitous enzymes which initiate the cleavage of extracellular glutathione (γ-Glu-Cys-Gly, GSH) into its constituent glutamate, cysteine, and glycine and catalyze the transfer of its γ-glutamyl group to water (hydrolysis), amino acids or small peptides (transpeptidation). These proteins utilize a conserved Thr residue to process their chains into a large and a small subunit that then form the catalytically competent enzyme. Multiple sequence alignments have shown that some bacterial γ-GTs, including that from Bacillus licheniformis (BlGT), possess an extra sequence at the C-terminal tail of the large subunit, whose role is unknown. Here, autoprocessing, structure, catalytic activity and stability against both temperature and the chemical denaturant guanidinium hydrochloride of six BlGT extra-sequence deletion mutants have been characterized by SDS-PAGE, circular dichroism, intrinsic fluorescence and homology modeling. Data suggest that the extra sequence has a crucial role in enzyme activation and structural stability. Our results assist in the development of a structure-based interpretation of the autoprocessing reaction of γ-GTs and are helpful to unveil the molecular bases of their structural stability.     

Inhibition of muscle actomyosin ATPase activity by mitochondrial ATPase inhibitor.

Ascites hepatoma cell line AH-130 was tested for the ability to transport various amino acids and glutathione before and after γ-glutamyl transpeptidase of the cells was affinity-labeled and inactivated by 6-diazo-5-oxo-L-norleucine, a glutamine analog. The rate of uptake of alanine, glycine, leucine and glutamine by the cells remained unchanged after γ-glutamyl transpeptidase was inactivated by this affinity label. This indicated that γ-glutamyl transpeptidase of the cell was not involved in the transport process of these amino acids tested. The uptake of glutathione was also tested before and after affinity labeling the enzyme. The total amount of the radioactivity incorporated into the cells was not significantly affected by the enzyme inactivation. However, the relative amount of incorporated intact glutathione was found to be slightly but significantly increased after membraneous γ-glutamyl transpeptidase was inactivated by the affinity label, while that of component amino acid, glycine, was found to decrease. This indicated that glutathione was taken up by the cell in its intact form as well as in degraded forms into its component amino acids, and γ-glutamyl transpeptidase in the ascites tumor cell AH-130 seemed to be involved in the metabolic process via the latter system.     

Specificity of a particulate rat renal peptidase and its localization along with other enzymes of mercapturic acid synthesis

Renal processing of S-derivatized glutathiones to mercapturic acids requires the participation of three enzymatic activities: γ-glutamyl hydrolase or transpeptidase, a peptidase which is capable of hydrolyzing S-derivatized cysteinylglycine, and an N-acetyltransferase. A particulate peptidase, which was assayed with S-benzylcysteine-p-nitroanilide, was found to be localized along with γ-glutamyltranspeptidase and N-acetyltransferase in the outer stripe region of the renal medulla. This localization suggests that these three activities may be contained primarily in the proximal straight tubules. Results of differential and isopycnic centrifugation indicate that the particulate peptidase is contained along with γ-glutamyltranspeptidase in the brush border membranes while the N-acetyltransferase is probably associated with the endoplasmic reticulum. The partially purified peptidase (200-fold) exhibits a broad substrate specificity. It has greater activity with reduced than oxidized cysteinylglycine, but S-derivatized substrates are hydrolyzed even faster. Comparison of its activity with various substrates indicates that it prefers peptides with a hydrophobic N-terminal amino acid and that it may require a free amino group. Heat-inactivation studies suggest that all of these activities are attributable to a single enzyme. These results suggest that this peptidase may participate along with γ-glutamyltranspeptidase and an N-acetyltransferase in the conversion of glutathione conjugates to mercapturic acids.     

Subcellular distribution of glutathione and cysteine in cyanobacteria

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria.     

Subcellular distribution of glutathione precursors in Arabidopsis thaliana

Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) - the first enzyme of glutathione synthesis - causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells.     

Overcoming the membrane barrier: Recruitment of γ-glutamyl transferase for intracellular release of metabolic cargo from peptide vectors

Semipermeable membranes of cells frequently pose an obstacle in metabolic engineering by limiting uptake of substrates, intermediates, or xenobiotics. Previous attempts to overcome this barrier relied on the promiscuous nature of peptide transport systems, but often suffered from low versatility or chemical instability. Here, we present an alternative strategy to transport cargo molecules across the inner membrane of Escherichia coli based on chemical synthesis of a stable cargo-peptide vector construct, transport through the peptide import system, and efficient intracellular release of the cargo by the promiscuous enzyme γ-glutamyl transferase (GGT). Retaining the otherwise periplasmic GGT in the cytoplasm was critical for the functionality of the system, as was fine-tuning its expression in order to minimize toxic effects associated to cytoplasmic GGT expression. Given the established protocols of peptide synthesis and the flexibility of peptide transport and GGT, the system is expected to be suitable for a broad range of cargoes.     

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.     

Purification and Properties of an Exocellular γ-Glutamyl Arylamidase from Bacillus sp. Strain No. 12

An exocellular γ-glutamyl arylamide-hydrolyzing enzyme was produced by a Bacillus sp. in L-glutamate-containing medium. This enzyme was a tetrameric simple protein composed of two heavy subunits (Mr 56,000) and two light subunits (Mr 46,000). It hydrolyzed γ-amido, acyl and aryl bonds in L- and D-glutamyl compounds, and the activity on L-glutamic acid γ-p-nitroanilide was inhibited by the addition of glutamate and γ-glutamyl compounds but not by α-glutamyl compounds. The activity was stimulated by various dipeptides but not by free amino acids, L-Alanine, glycine, L-serine and L-cysteine inhibited the enzyme competitively. Addition of hy-droxylamine had no effect on the activity.     

A two-stage glycine supplementation strategy enhances the extracellular expression of sortase A in Escherichia coli

Sortase A (SrtA) is a transpeptidase widely used in protein engineering. In this study, the enhancement of the extracellular expression of SrtA in Escherichia coli was investigated using a combined strategy based on the PelB signal peptide and chemical additives. First, glycine was identified to be the best additive for promoting the release of SrtA from the periplasm to the medium. Then, the effect of glycine concentration on cell growth and SrtA production was investigated, and a two-stage supplementation strategy was developed in order to control the impairment of cell growth and to achieve the maximum production of secretory SrtA. The results showed that when 0.5% glycine was added to the medium at the beginning of cell growth and 1% glycine was added at the end of the exponential phase, the extracellular yield of SrtA was 228.0 mg/L and the enzyme activity was 100.4 U/mL at the end of fermentation; these values were 5.3- and 8.6-fold higher, respectively, than those attained in the control culture without any additives. This result represents the highest yield of extracellular SrtA ever reported and demonstrates a promising process for the production of SrtA for large-scale industrial application.     

Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: An enzyme specialized in hydrolase activity

Gamma-glutamyltranspeptidases (γ-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, γ-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce γ-glutamyl compounds. To find out γ-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. γ-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using l-glutamic acid γ-(4-nitroanilide) as the substrate reveals that the active enzyme has K_m 7.6 μM and V_max 0.36 μmol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 °C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other γ-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized γ-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability.     

Enzymatic synthesis of theanine with Escherichia coli γ-glutamyltranspeptidase from a series of γ-glutamyl anilide substrate analogues

In order to investigate the catalytic mechanism of Escherichia coli γ-glutamyltranspeptidase, ten para- and meta-substituted γ-glutamyl anilides were chemically prepared and employed as substrates to synthesize L-theanine to assay the activity of γ-glutamyltranspeptidase. The reaction was optimized for γ-glutamyl-p-nitroanilide. Key factors such as substrate specificity, pH, temperature, and the substrate mole ratio were all investigated. Kinetic studies of the acyl transfer reaction were described and the Hammett plot was constructed. This study indicated that the ratelimiting acylation reaction of γ-glutamyltranspeptidase can apparently be accelerated by either the electron-withdrawing or electron-donating substituents of γ-glutamyl anilides. The reaction could be catalyzed by the general acid and carboxy of Asp-433 or phenolic hydroxyl Tyr-444 may be the acid by autodock simulation for all prepared γ-glutamyl anilides.     

Nonidentity of sulfhydryl oxidase and gama-glutamyltransferase in bovine milk

Sulfhydryl oxidase (glutathione-oxidizing activity) is closely associated with γ-glutamyltransferase (γ-glutamyl transpeptidase) in skim milk membranes. Similar close association of the two enzymatic activities in kidney membranes has led to the recent proposal that glutathione-oxidizing activity can be attributed to the action of γ-glutamyltransferase, itself, in generating cysteinylglycine which, in turn, catalyzes sulfhydryl group oxidation (O. W. Griffith and S. S. Tate, 1980, J. Biol. Chem.255, 5011–5014). However, a previously published procedure for the isolation of highly purified sulfhydryl oxidase from skim milk membranes (V. G. Janolino and H. E. Swaisgood, 1975, J. Biol. Chem.250, 2532–2538) leads to the effective separation of the two activities. Quantitative chromatographic analyses of GSH, GSSG, and Glu levels revealed that the highly purified sulfhydryl oxidase preparation catalyzes the direct oxidation of GSH to GSSG without detectable cleavage of the γ-glutamyl peptide bond. These results were confirmed by monitoring the time course of substrate disappearance and product formation using high-performance liquid chromatography. Conversely, a supernatant fraction enriched in γ-glutamyltransferase activity displayed no sulfhydryl group-oxidizing activity. 6-Diazo-5-oxo-l-norleucine selectively inhibited the transferase in crude preparations containing both sulfhydryl oxidase and γ-glutamyltransferase. It is concluded that sulfhydryl oxidase and γ-glutamyltransferase activities are distinct and separable.     

Production of thermophilic α-amylase using immobilized transformed Escherichia coli by addition of glycine

Efficient production of thermophilic α-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with κ-carrageenan by the addition of glycine. The effects of glycine, the concentrations of κ-carrageenan and KCI on the production of the enzyme as well as the stability of plasmid pHI301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of α-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term “release”, while diffusion-out from gel beads is referred to by the term “liberate”.) Concentrations of KCI and immobilizing support significantly affected on the liberation of α-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCI, which is a hardening agent for carrageenan. Although the viability of cells and α-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of α-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.     

Enzyme organization in the proline biosynthetic pathway of Escherichia coli

The conversion of glutamic acid to proline by an Escherichia coli extract was studied. The activity was dependent upon the presence of ATP and NADPH and was largely unaffected by the presence of NH₃ or imidazole. The first two pathway enzymes appear to exist as a complex which stabilizes a labile intermediate postulated as γ-glutamyl phosphate. Attempted synthesis of this compound was unsuccessful due to its spontaneous cyclization to 2-pyrrolidone 5-carboxylate. Dissociation of the enzyme complex upon dilution of the extract is presumed responsible for an experimentally observed “dilution effect”. E. coli pro_A^? and pro_B^? auxotroph extracts failed to complement one another in the biosynthesis of proline. This is attributed to the lack of a dynamic equilibrium between the complex and its constituent enzymes.In vivo studies with E. coli showed no evidence for metabolic channeling in the final reaction of proline synthesis, the reduction of Δ¹-pyrroline 5-carboxylate.     

Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization,yield and activity

Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest.