A novel catalytic ability of gamma-glutamylcysteine synthetase of Escherichia coli and its application in theanine production.

Gamma-glutamylcysteine synthetase (gammaGCS, EC 6.3.2.2) catalyzes the formation of gamma-glutamylcysteine from L-glutamic acid (Glu) and L-cysteine (Cys) in an ATP-dependent manner. While gammaGCS 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 gammaGCS from Escherichia coli has the ability to combine Glu and amines to form gamma-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 gammaGCS was used in the production of theanine (gamma-glutamylethylamine). The resting cells of E. coli expressing gammaGCS, in which ATP was regenerated through glycolysis, synthesized 12.1 mM theanine (18 h) from 429 mM ethylamine.

Gamma-glutamylcysteine synthetase from Plasmodium berghei.

Gamma-glutamylcysteine synthetase (L-glutamate-L-cysteine ligase, gamma-GCS, EC 6.3.2.2.), the rate limiting enzyme in glutathione biosynthetic pathway has been analysed in the asexual erythrocytic stages of rodent malaria parasite, Plasmodium berghei and its host erythrocytes. Cell-free parasite isolated by saponin lysis contained about 2 and 8 times higher activity of gamma-GCS compared to P. berghei-infected and normal mice erythrocytes respectively. Subcellular fractionation revealed that the enzyme was mainly confined to the cytosolic part of the parasite. gamma-GCS from P. berghei was purified employing ammonium sulphate precipitation, Sephadex G-200 gel filtration and anionic exchange chromatography on DEAE-cellulose. There was 51.6 fold purification of enzyme and its specific activity was 39.5 U/mg. SDS-PAGE showed P. berghei gamma-GCS as a heterodimer dissociating into two non-identical sub-units of 66 kDa and 57 kDa. The enzyme was observed as white band of activity on native polyacrylamide gel stained for specific gamma-GCS activity. Km values for L-Cys, ATP and L-Glu were 0.53 mM, 0.92 mM and 0.75 mM, respectively. The inhibition of gamma-GCS activity by glutathione was found to be competitive with respect to glutamate (Ki=1.53 mM) and non competitive to ATP and cysteine. Antimalarial drugs did not show any significant effect on parasite gamma-GCS. Parasite enzyme induced humoral response in mice demonstrated by ELISA, IFA and immunoblotting and exhibited partial protection against P. berghei infection suggesting a significant role of P. berghei gamma-GCS in malaria control.

Over expression of glutamate cysteine ligase increases cellular resistance to H2O2-induced DNA single-strand breaks.

Hydrogen peroxide (H2O2) can cause single strand DNA breaks (ssDNA) in cells when the mechanisms normally in place to reduce it are overwhelmed. Such mechanisms include catalase, glutathione peroxidases (GPx), and peroxiredoxins. The relative importance of these enzymes in H2O2 reduction varies with cell and tissue type. The role of the GPx cofactor glutathione (GSH) in oxidative defense can be further understood by modulating its synthesis. The first and rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which has a catalytic subunit (Gclc) and a modifier subunit (Gclm). Using mouse hepatoma cells we evaluated the effects of GCL over expression on H2O2-induced changes in GSH and ssDNA break formation with the single cell gel electrophoresis assay (SCG or comet assay), and the acridine orange DNA unwinding flow cytometry assay (AO unwinding assay). Cells over expressing GCL had higher GSH content than control cells, and both SCG and AO unwinding assays revealed that cells over expressing GCL were significantly more resistant to H2O2-induced ssDNA break formation. Furthermore, using the AO unwinding assay, the prevalence of H2O2-induced breaks in different phases of the cell cycle was not different, and the degree of protection afforded by GCL over expression was also not cell cycle phase dependent. Our results support the hypothesis that GCL over expression enhanced GSH biosynthesis and protected cells from H2O2-induced DNA breaks. These results also suggest that genetic polymorphisms that affect GCL expression may be important determinants of oxidative DNA damage and cancer.

Time-course development of the Cd2+ hyper-accumulating phenotype in Euglena gracilis.

To determine the onset of the Cd2+-hyperaccumulating phenotype in Euglena gracilis, induced by Hg2+ pretreatment (Avilés et al. in Arch Microbiol 180:1-10, 2003), the changes in cellular growth, Cd2+ uptake, and intracellular contents of sulfide, cysteine, gamma-glutamylcysteine, glutathione and phytochelatins during the progress of the culture were analyzed. In cells exposed to 0.2 mM CdCl2, the Cd2+-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth and higher internal contents of sulfide and thiol-compounds, along with a higher gamma-glutamylcysteine synthetase activity. However, the stiochiometry of thiol-compounds/Cd2+ accumulated was similar for both control and Hg2+-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully inactivate Cd2+. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the Cd2+-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd2+ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd2+ compartmentation into organelles.

YbdK is a carboxylate-amine ligase with a gamma-glutamyl:Cysteine ligase activity: crystal structure and enzymatic assays.

The Escherichia coli open reading frame YbdK encodes a member of a large bacterial protein family of unknown biological function. The sequences within this family are remotely related to the sequence of gamma-glutamate-cysteine ligase (gamma-GCS), an enzyme in the glutathione biosynthetic pathway. A gene encoding gamma-GCS in E. coli is already known. The 2.15 A resolution crystal structure of YbdK reveals an overall fold similar to that of glutamine synthetase (GS), a nitrogen metabolism enzyme that ligates glutamate and ammonia to yield glutamine. GS and gamma-GCS perform related chemical reactions and require ATP and Mg2+ for their activity. The Mg2+-dependent binding of ATP to YbdK was confirmed by fluorescence spectroscopy employing 2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate, and yielding a dissociation constant of 3 +/- 0.5 microM. The structure of YbdK contains a crevice that corresponds to the binding sites of ATP, Mg2+ and glutamate in GS. Many of the GS residues that coordinate the metal ions and interact with glutamic acid and the phosphoryl and ribosyl groups of ATP are also present in YbdK. GS amino acids that have been associated with ammonia binding have no obvious counterparts in YbdK, consistent with a substrate specificity that is different from that of GS. Ligase activity between glutamic acid and each of the twenty amino acid residues was tested on high performance liquid chromatography (HPLC) by following the hydrolysis of ATP to ADP. Catalysis was observed only with cysteine. A pyruvate kinase/lactic acid dehydrogenase coupled assay was used to rule out GS activity and to determine that YbdK exhibits gamma-GCS activity. The catalytic rate was found to be approximately 500-fold slower than that reported for authentic gamma-GCS.

Escherichia coli B gamma-glutamylcysteine synthetase: modification, purification, crystallization and preliminary crystallographic analysis.

Escherichia coli B gamma-glutamylcysteine synthetase (gammaGCS) catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form the glutathione precursor gamma-L-Glu-Cys and is a target for development of potential therapeutic agents. By introducing four point mutations of surface-exposed cysteine residues to serine, the gammaGCS was purified to homogeneity; single crystals have been obtained using the hanging-drop vapour-diffusion method with sodium formate. The gammaGCS crystal diffracted to 2.8 A and belongs to space group R3, with unit-cell parameters a = b = 326.7, c = 103.9 A.

The enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase.

The metabolite glutathione fulfills many important and chemically complex roles in protecting cellular components from the deleterious effects of toxic species. GSH combines with hydroxyl radical, peroxynitrite, and hydroperoxides, as well as reactive electrophiles, including activated phosphoramide mustard. This thiol-containing reductant also maintains so-called thiol-enzymes in their catalytically active form, and maintains vitamins C and E in their biologically active forms. The key step in glutathione synthesis, namely the ATP-dependent synthesis of gamma-glutamylcysteine, is the topic of this review. Details are presented on (a) the enzyme's purification and protein chemistry, (b) the successful cDNA cloning, and characterization of the genes responsible for the biosynthesis of this enzyme. After considering aspects of the role of overexpression of this synthetase in terms of cancer chemotherapy, attention is focused on post-translational regulation. The remainder of the review deals with the catalytic mechanism (including substrate specificity, reactions catalyzed, steady-state kinetics, and chemical mechanism) as well as the inhibition of the enzyme (via feedback inhibition, reaction with S-alkyl homocysteine sulfoximine inhibitors, the clinical use of buthionine sulfoximine with cancer patients, and inactivation by cystamine, chloroketones, and various nitric oxide donors).

Expression and purification of human gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the ATP-dependent ligation of L-glutamate and L-cysteine to form L-gamma-glutamyl-L-cysteine; this is the first and rate-limiting step in glutathione biosynthesis. Inhibitors of gamma-GCS such as buthionine sulfoximine are widely used as tools for elucidating glutathione metabolism in vivo and as pharmacological agents for reversing glutathione-based resistance to chemotherapy and radiation therapy in certain cancers. Although gamma-GCS is readily isolated from rat kidneys, future drug design efforts are better based on structure-activity relationships established with the human enzyme. We report here the coexpression in Escherichia coli BL21(DE3) of the human gamma-GCS catalytic (heavy) subunit and regulatory (light) subunit using pET-3d and pET-9d vectors, respectively. Intracellular assembly of the holoenzyme occurred without difficulty, and levels of expression were acceptable (approximately 32 mg holoenzyme/100 g cells). Recombinant human gamma-GCS was purified to homogeneity in an overall yield of 45% by ammonium sulfate fractionation followed by sequential chromatography on Q-Sepharose ion-exchange, Superdex 200 gel filtration and ATP-affinity resins. Trace amounts of E. coli gamma-GCS were removed by immunoaffinity chromatography. The specific activity of the isolated enzyme was >1500 units/mg, comparable to the best preparations from rat kidney. The Km values for L-glutamate, L-cysteine, L-gamma-aminobutyrate (an L-cysteine surrogate), and ATP are 1.8, 0.1, 1.3, and 0.4 mM, respectively. Recombinant human gamma-GCS, like native rat gamma-GCS, is feedback inhibited by glutathione and is potently inhibited by buthionine sulfoximine and cystamine.

Characterisation of gamma-glutamylcysteine synthetase-heavy subunit promoter: a critical role for AP-1.

The 5'-flanking region of human gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) was characterised by creating a series of chloramphenicol acetyl transferase (CAT) reporter deletion constructs. Analysis of various deleted CAT constructs revealed that a putative AP-1 consensus sequence is required to direct the constitutive and oxidant-mediated promoter activity. Gel mobility shift and mutation analysis of the sequence (-269 to -263 bp), showed binding of AP-1 is involved in the oxidant-mediated regulation of gamma-GCS-HS promoter activity.

Regulation of human gamma-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells.

We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.

Regulation of gamma-glutamylcysteine synthetase by protein phosphorylation.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited approximately 20-40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg(2+)-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its K(m). Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.

Purification and characterization of gamma-glutamylcysteine synthetase from Ascaris suum.

We have purified and characterized the Ascaris suum gamma-glutamylcysteine synthetase, the rate-limiting step in the glutathione biosynthesis. The purified enzyme exhibited a specific activity of 18 U (mg protein)-1. Estimation of the molecular mass of the native enzyme by FPLC on Superdex S-200 revealed the presence of two enzyme activity peaks corresponding to molecular masses of 100 and 70 kDa. The higher-molecular-mass component could be dissociated by repeated gel filtration into the 70-kDa protein which is the enzymatically active subunit. The apparent Km values of the A. suum enzyme for L-aminobutyrate, L-cysteine and L-glutamate were 0.31, 0.41 and 0.94 mM, respectively. D,L-Buthionine-S,R-sulfoximine and cystamine showed time-dependent irreversible inhibitory effects on the A. suum enzyme activity with Ki values of 0.05 and 1.11 microM, respectively. The Ki values for the corresponding enzyme from rat kidney with D,L-buthionine-S,R-sulfoximine and cystamine were 7.19 and 22.2 microM, respectively. The time of half-inactivation of the enzyme at infinite concentration of D,L-buthionine-S,R-sulfoximine, tau 50, was determined to be 3.1 and 1.34 min, for the parasite and mammalian enzymes respectively. For cystamine, a tau 50 value of 3.32 min for the A. suum gamma-glutamylcysteine synthetase was determined, while a value of 2 min in case of rat kidney enzyme was found. The A. suum enzyme activity was competitively inhibited by glutathione with a Ki value of 0.11 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical regulation of the activity of gamma-glutamylcysteine synthetase from rat liver and kidney by glutathione.

Rat liver and kidney gamma-glutamylcysteine synthetase (gamma GCS) had similar catalytic properties and consisted of heavy and light subunits, but the molecular structure of the two enzymes was not the same as evidenced by the results of SDS-PAGE and disc gel electrophoresis. Unlike kidney enzyme, most of liver gamma GCS was in a reduced enzyme form which did not have disulfide linkage between heavy and light subunits. Although the oxidized form of the two enzymes which subunits were linked with disulfide bond(s) could be dissociated to a similar extent by GSH, liver gamma GCS was inhibited by GSH to a much greater extent. These results suggest that the relative sensitivity of the gamma GCS enzymes to inhibition by GSH might be related to the inherent dissociability of heavy and light subunit of gamma GCS.

Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase (rat kidney), which catalyzes the first step of GSH synthesis, can be dissociated into subunits (M(r) 73,000 and 27,700) by native gel electrophoresis after treatment with dithiothreitol (DTT); the heavy subunit, which exhibits catalytic activity and feedback inhibition by GSH (Seelig, G. F., Simondsen, R. P., and Meister, A. (1984) J. Biol. Chem. 259, 9345-9347), was cloned and sequenced (Yan, N., and Meister, A. (1990) J. Biol. Chem. 265, 1588-1593). Here, the cDNA for the heavy sub unit was expressed in Escherichia coli, and the recombinant enzyme was separated from E. coli gamma-glutamylcysteine synthetase and purified. The recombinant enzyme and the isolated heavy subunit have much lower affinity for glutamate and higher sensitivity to GSH inhibition than the holoenzyme, suggesting that the heavy subunit alone would not be very active in vivo. A GSH analog, gamma-Glu-alpha-aminobutyryl-Gly (ophthalmic acid), inhibits only slightly, but inhibits much more after treatment of the holoenzyme with DTT. In contrast, ophthalmic acid inhibits the recombinant and isolated heavy subunit enzymes substantially without DTT treatment. We conclude that (a) the light subunit has a regulatory function affecting the affinity of the enzyme for glutamate and GSH and (b) feedback inhibition by GSH involves reduction of the enzyme and also competition between GSH and glutamate for the glutamate site.

gamma-Glutamylcysteine synthetase and active transport of glutathione S-conjugate are responsive to heat shock in K562 erythroid cells.

Effect of heat shock on a glutathione-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and ATP-dependent outward transport of glutathione S-conjugate was characterized using K562 erythroid cells. When K562 cells grown at 37 degrees C were shifted to 42 degrees C for 2 h, an approximate 1.7-fold increase in the activity of gamma-GCS was observed. Treatment of K562 cells with erythropoietin (EP) for 12 h resulted in a decrease in the activity of gamma-GCS to 64% of the control. However, responsiveness of this enzyme activity in the cells treated with EP to heat shock was similar to that in untreated cells. Changes in the immunological activity of gamma-GCS were also observed in parallel with those in the enzymatic activity. On Northern blot analysis of total RNAs isolated from the cells with human cDNA for gamma-GCS, a substantial induction of mRNA level was found by heat shock and a reduction of EP. These changes were modest but correlated to the mRNA expression of a heat shock protein, HSP 70. Heat shock also had an effect of 1.8-fold stimulation on glutathione S-conjugate transport in K562 cells previously incubated with 1-chloro-2,4-dinitrobenzene. Treatment of the cells with EP resulted in a decrease in this transport by 62%. Similarly, the levels of glutathione S-conjugate-stimulated Mg(2+)-ATPase, which enzyme is thought to be involved in the transport of glutathione S-conjugate, were responsive to heat shock and EP. These results suggest that glutathione synthesis and transport process of glutathione metabolites are responsive to heat shock and play a role in the defense system against stresses. It is also suggested that the regulatory site of the expression of these enzymes by heat shock is independent of that by EP.

Amino acid sequence and function of the light subunit of rat kidney gamma-glutamylcysteine synthetase.

The heavy subunit (M(r), 72,614) of rat kidney gamma-glutamylcysteine synthetase, the enzyme that catalyzes the first step of glutathione (GSH) synthesis, mediates the catalytic activity of this enzyme and its feedback inhibition by GSH. There is evidence that the light subunit has a regulatory function (Huang, C.-S., Chang, L.-S., Anderson, M.E., and Meister, A. (1993) J. Biol. Chem. 268, 19675-19680). In the present work the cDNA for the light subunit was isolated, sequenced, and expressed in Escherichia coli. The cDNA was found to code for a protein of 274 amino acid residues (M(r) 30, 548). Recombinant holoenzyme was obtained by co-expression of the heavy and light subunits and by mixing of the separately expressed proteins. These recombinant holoenzyme preparations exhibit catalytic and GSH feedback inhibitory properties that are virtually identical to those of the isolated holoenzyme. These studies establish that the light subunit is an integral part of the enzyme and that the light and heavy subunits, are coded for separately. Possibly significant similarity of sequence of amino acids was found between the light subunit and E. coli gamma-glutamylcysteine synthetase, which is a single polypeptide.

Purification and biochemical characterization of gamma-glutamylcysteine synthetase from a human malignant astrocytoma cell line.

gamma-Glutamylcysteine synthetase (EC 6.3.2.2.) the key regulatory enzyme in glutathione biosynthesis was purified from a human malignant astrocytoma cell line using a combination of ammonium sulfate fractionation, DE-52 cellulose chromatography and ATP-agarose affinity chromatography. The purified protein had a specific activity of 1725 units/mg protein, which represented an 86-fold purification and a 22% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major subunits with apparent molecular sizes of 72 kDa and 32 kDa. The Km values for L-glutamate and L-alpha-aminobutyrate were 0.03 mM and 0.14 mM respectively. These molecular and catalytic properties of gamma-glutamylcysteine synthetase from astrocytoma cell line are similar, but not identical to those purified from rat kidney.

Structural studies on the inactivation of gamma-glutamylcysteine synthetase by the disulphide analogues of radioprotective cysteamine derivatives. Effects of aminoalkyl and hydroxyalkyl chain length and beta beta-bis-dimethylation.

Disulphide compounds have been shown to inactivate gamma-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis. Such compounds bind to a cysteine residue at or near the glutamate-binding site of the enzyme. This phenomenon is thought to be responsible for the synergistic toxicity of the thiophosphate radio- and chemo-protective agent WR2721 and the oxygen-radical generator 6-hydroxydopamine (2,4,6-trihydroxyphenethylamine). 6-Hydroxy-dopamine enhances conversion of WR2721 into its disulphide metabolite NN'-bis-(3-aminopropyl)cystamine, which, in turn, paralyses the synthetase. In an effort to identify radio- and chemo-protective thiols and thiol derivatives that do not have this toxicity, we have begun to define the structure-activity relationship that governs inactivation of the enzyme by analogues of WR2721 disulphide. NN'-Bis(aminoalkyl)cystamines and bis(hydroxyalkyl)cystamines with an alkyl chain length of C5 or greater are not inactivators of the synthetase. That this is not due solely to the size of these compounds is shown by the potent inactivation of the enzyme by SAPH3 disulphide, an extremely bulky cystamine analogue. beta beta-Bis-dimethylation of the cystamine portion of the molecule also obviates inactivation. This is almost certainly due to steric interference with disulphide interchange. These findings may facilitate the safe adjunctive use of the thiol counterparts of such compounds with oxygen-radical-generating chemotherapeutic agents, and may shed light on the structure of the region of the synthetase adjacent to the glutamate-binding site.