Enzymatic synthesis of γ-l-glutamyl-S-allyl-l-cysteine,a naturally occurring organosulfur compound from garlic,by Bacillus licheniformis γ-glutamyltranspeptidase

In the practical application of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT), we describe a straightforward enzymatic synthesis of γ-L-glutamyl-S-allyl-L-cysteine (GSAC), a naturally occurring organosulfur compound found in garlic, based on a transpeptidation reaction involving glutamine as the γ-glutamyl donor and S-allyl-L-cysteine as the acceptor. With the help of thin layer chromatography technique and computer-assisted image analysis, we performed the quantitative determination of GSAC. The optimum conditions for a biocatalyzed synthesis of GSAC were 200 mM glutamine, 200 mM S-allyl-L-cysteine, 50 mM Tris–HCl buffer (pH 9.0), and BlGGT at a final concentration of 1.0 U/mL. After a 15-h incubation of the reaction mixture at 60 °C, the GSAC yield for the free and immobilized enzymes was 19.3% and 18.3%, respectively. The enzymatic synthesis of GSAC was repeated under optimal conditions at 1-mmol preparative level. The reaction products together with the commercially available GSAC were further subjected to an ESI-MS/MS analysis. A significant signal with m/z of 291.1 and the protonated fragments at m/z of 73.0, 130.1, 145.0, and 162.1 were observed in the positive ESI-MS/MS spectrum, which is consistent with those of the standard compound. These results confirm the successful synthesis of GSAC from glutamine and S-allyl-L-cysteine by BlGGT.     

Molecular and kinetic properties of purified γ-glutamyl transpeptidase from yeast (Saccharomyces cerevisiae)

The enzyme γ-glutamyl transpeptidase was purified from the yeast Saccharomyces cerevisiae by a procedure involving protamine sulfate treatment, chromatography on DEAE-Sephadex A 50, salt fractionation, successive chromatography on Sephadex G 150 and lentil lectin sepharose 6B. The procedure achieves 25 % yield and 4200-fold purification. The final preparation is a glycoprotein (M, 90 000) containing 31.4 % carbohydrates and composed of two non-identical subunits (M, 64 000 and 23 000). The specificity patterns of the yeast enzyme are rather similar to those of mammalian and higher plant transpeptidases. The enzyme mechanism might be of the double displacement (ping-pong) type.     

Lack of correlation between glutathione turnover and amino acid absorption by the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae was grown in the presence of 1.0 mM l-methionine and the half-life of degradation of glutathione determined for the strains Σ1278b (444 min) and the amino-acid-uptake deficient mutant 2512c (368 min). There is no significant difference in these values, yet the rate of uptake of l-methionine is 5–7 times lower in the mutant. In neither strain is the turnover of glutathione sufficient to account for amino acid uptake. We conclude that there is no correlation between the γ-glutamyl cycle and amino acid uptake by this east.     

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.     

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0–32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.     

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.     

Inhibitory effects in vitro of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-glutathione,a proposed metabolite of l-histidine,on γ-glutamyltransferase activity

A transfer of the γ-glutamyl moiety of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (I), an adduct of glutathione and l-histidine metabolite urocanic acid, has been investigated by using γ-glutamyltransferase preparation from bovine kidney. When an equimolar mixture of two diastereomers of compound I in a phosphate buffer was allowed to react with glycylglycine in the presence of the transferase, two diastereomers of N-{S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteinyl}glycine (II) were formed in the same yield with each other and this was accompanied by a formation of γ-glutamylglycylglycine. Kinetics of compound I with the transferase indicated high affinity between the two materials, while the maximal reaction velocity of the γ-glutamyl transfer was low. Effects of compound I in vitro on the transfer of γ-glutamyl moiety of γ-glutamyl-p-nitroanilide to glycylglycine with the transferase were also studied, and the results indicated that the transfer was suppressed by compound I based on its competitive and non-competitive inhibitions. These results suggest that little variation in reactivities of two diastereomers of compound I as the substrate is given by the difference in stereomerism of their asymmetric carbon atoms and that inhibitory effects of compound I on the catalytic action of the transferase is of sufficient physiological importance to decrease the degradation of natural γ-glutamyl compounds, such as glutathione and its analogs.     

Synthesis of the Enantiomers of (Z)-5-(1-Octenyl)oxacyclopentan-2-one,a Sex Pheromone of the Cupreous Chafer Beetle,Anomala cuprea Hope

Glutaminase (EC 3.5.1.2) was isolated from Pseudomonas nitroreducens IFO 12694 grown on 0.6% sodium glutamate as a nitrogen source (325-fold purification, 13% yield). The molecular weight of the enzyme was estimated to be 40,000 by gel filtration and SDS-gel electrophoresis. The enzyme hydro-lyzed glutamine optimally at pH 9, and its K_m was 6.5 mm. d-Glutamine, γ-glutamyl p-nitroanilide, γ-glutamylmethylamide, γ-glutamylethylamide (theanine), and glutathione showed respectively 107, 85, 78, 74, and 82% reactivity of glutamine. Zn²⁺, Ni²⁺, Cd²⁺, Co²⁺, Fe²⁺, and Cu²⁺ repressed the enzyme activity strongly.

Glutaminase formed γ-glutamylhydroxamate in the reaction mixture containing glutamine and hydroxylamine (transferring reaction). The optimum pH of the transferring reaction was 7–8, and the K_m for glutamine and hydroxylamine were 4 mm and 120 mm, respectively. γ-Glutamyl derivatives hydrolyzable by glutaminase showed reactivity for the transferring reaction. Methylamine or ethylamine was replaceable for hydroxylamine with 3 or 8% reactivity. The effect of divalent cations was not so striking as in the hydrolyzing reaction.     

γ-l-Glutamyl-l-pipecolic acid in Gleditsia caspica

y-l-Glutamyl-l-pipecolic acid has been isolated from seeds of Gleditsia caspica (L.) Desf. Proof of its structure was obtained by chromatographic and spectroscopic examination of the natural product and its hydrolytic products. The new compound is the first example of a naturally occurring γ-glutamyl imino acid.     

Diversity of γ- glutamyl peptides and oligosaccharides,the “kokumi” taste enhancers,in seeds from soybean mini core collections

Soybeans (Glycine max (L,) Merr,) contain γ-glutamyl peptides and oligosaccharides, and these components play an important role in imparting the “kokumi” taste to foods. To gain insight into the genetic diversities and molecular mechanisms of accumulation of γ-glutamyl peptides and oligosaccharides in soybean, we measured the contents of these components using the Japan and World mini core collections. Similar to other previously reported traits, wide variations were detected among the accessions in the core collections with respect to the content of γ-glutamyl peptides and oligosaccharides. We found a positive relationship between the content of γ-glutamyl tyrosine and γ-glutamyl phenylalanine and between the content of raffinose and stachyose. Furthermore, there were unique accessions that included high levels of γ-glutamyl peptides and oligosaccharides. These accessions may be helpful in understanding the accumulation mechanism of γ-glutamyl peptides and oligosaccharides and to increase the “kokumi” taste components in soybean by performing a genetic analysis.     

γ-Glutamyltranspeptidases in the metabolism of γ-glutamyl peptides in plants

The activity and specificity of γ-glutamyltranspeptidase in immature seeds of some leguminous plants did not reflect the γ-glutamyl peptide pattern     

Structure of the Hydrolyzed Product (F-2) Released from γ-Polyglutamic Acid by γ-Glutamyl Hydrolase YwtD of Bacillus subtilis

The structure of the hydrolyzed product (F-2) with a molecular mass of about 2 kDa released from γ-polyglutamic acid by the γ-glutamyl hydrolase YwtD of Bacillus subtilis was analyzed. The results showed that F-2 is an optically heterogeneous polymer consisting of D- and L-glutamic acid in an 80:20 ratio with D-glutamic acid on both the N- and C-terminal sides, suggesting that YwtD is an enzyme that cleaves the γ-glutamyl bond between D- and D-glutamic acid recognizing adjacent L-glutamic acid toward the N-terminal region.     

A kinetic study of gamma-glutamyltransferase (GGT)-mediated S-nitrosoglutathione catabolism

S-Nitrosoglutathione (GSNO) is a nitric oxide (NO) donor compound which has been postulated to be involved in transport of NO in vivo. It is known that γ-glutamyl transpeptidase (GGT) is one of the enzymes involved in the enzyme-mediated decomposition of GSNO, but no kinetics studies of the reaction GSNO-GGT are reported in literature.In this study we directly investigated the kinetics of GGT with respect to GSNO as a substrate and glycyl-glycine (GG) as acceptor co-substrate by spectrophotometry at 334 nm. GGT hydrolyses the γ-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and γ-glutamyl-GG. However, as both the substrate GSNO and the first product CGNO absorb at 334 nm, we optimized an ancillary reaction coupled to the enzymatic reaction, based on the copper-mediated decomposition of CGNO yielding oxidized cysteinyl-glycine and NO. The ancillary reaction allowed us to study directly the GSNO/GGT kinetics by following the decrease of the characteristic absorbance of nitrosothiols at 334 nm. A K_m of GGT for GSNO of 0.398 ± 31 mM was thus found, comparable with K_m values reported for other γ-glutamyl substrates of GGT.     

γ-Glutamyl transpeptidase of the ackee plant

The enzyme γ-glutamyl transpeptidase was purified from seeds of immature ackee fruit (Blighia sapida; Sapindaceae) by salt fractionation and gel filtration on Biogel P-10 and P-200. The procedure, which differs from an earlier one applied to kidney bean fruit, achieves 9.8% yield and 577-fold purification. The enzyme is also present in other parts of the fruit and in leaves. A MW of 12 500 was found by SDS-polyacrylamide gel electrophoresis, a value much lower that that reported for the enzyme from kidney bean fruit. Neutral or amino sugar accounts for 10% of the dry weight. In vitro, the enzyme catalysed synthesis of an unusual γ-glutamyl dipeptide which occurs in ackee seeds, using glutathione as glutamyl group donor. The enzyme mechanism was of the double displacement (ping-pong) type.     

Modification of the acceptor binding site of gamma-glutamyl transpeptidase by the diazonium derivatives of p-aminohippurate and p-aminobenzoate

Hippurate and maleate have been shown to bind to the aminoacylglycine (acceptor) binding site of γ-glutamyl transpeptidase, thereby stimulating the hydrolysis of γ-glutamyl compounds at the expense of transpeptidation (Thompson, G. A., and Meister, A. (1979) J. Biol. Chem.254, 2956–2960; Thompson, G. A., and Meister, A. (1980) J. Biol. Chem.255, 2109–2113). It has now been found that a number of benzoate derivatives also bind and modulate rat kidney transpeptidase, as indicated by their ability to enhance the rate of inactivation of transpeptidase by the glutamine antagonist l-(αS, 5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Furthermore, rapid loss of transpeptidase activity results upon preincubation of the enzyme with the diazonium derivatives of p-aminohippurate and p-aminobenzoate. The modified enzyme can still hydrolyze γ-glutamyl substrates but is no longer modulated by hippurate and maleate. Loss of transpeptidase activity was not associated with incorporation of radioactive label from diazotized [¹⁴C]p-aminohippurate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the modified enzyme revealed a nondissociable species, M_r 68,000, shown to result from crosslinking of the two subunits of transpeptidase (M_r 46,000 and 22,000, respectively). The crosslinking of the subunits paralleled the extent of inactivation of transpeptidation activity and both crosslinking and inactivation were prevented by treatment with the diazotized derivatives in the presence of either hippurate or maleate. These and other data indicate that the diazonium derivatives of p-aminohippurate and p-aminobenzoate interact with the acceptor binding site and produce a stable bond between amino acid residues in the vicinity of this site which, thus, appears to be located in the intersubunit contact region.     

Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active site cleft and a cluster of acid residues potentially involved in the recognition of a metal ion

γ-Glutamyltranspeptidases (γ-GTs) cleave the γ-glutamyl amide bond of glutathione and transfer the released γ-glutamyl group to water (hydrolysis) or acceptor amino acids (transpeptidation). These ubiquitous enzymes play a key role in the biosynthesis and degradation of glutathione, and in xenobiotic detoxification.     

Free amino acids and γ-glutamyl peptides in fagaceae

Amino acids have been investigated in seeds and fresh parts of members of the Fagaceae. Seeds from the genus Fagus contain willardiine, 5-hydroxy-6-methylpipecolic acids, N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]azetidine-2-carboxylic acid and γ-glutamyl peptides, mainly γ-glutamylphenylalanine. These compounds are nearly or totally absent from leaves of F. silvatica and from seedlings and immature seeds of F. silvatica var. purpurea; instead, the seedlings contain large amounts of γ-l-glutamyl-l-isoleucine and γ-l-glutamyl-l-leucine. γ-l-Glutamyl-l-tryptophan and γ-l-glutamyl-γ-l-glutamyl-l-phenylalanine, not previously known from nature, have been isolated from seeds of F. silvatica var. purpurea. The structures have been confirmed by syntheses. 4-Hydroxypipecolic acid (with trans-configuration) has been identified in seeds of F. japonica Maxim. and F. sieboldii Endl. None of the above compounds was found in Quercus or Castanea species whereas argininosuccinic acid was identified in Castanea sativa.     

Mathematically combined half-cell reduction potentials of low-molecular-weight thiols as markers of seed ageing

Abstract

The half-cell reduction potential of the glutathione disulphide (GSSG)/glutathione (GSH) redox couple appears to correlate with cell viability and has been proposed to be a marker of seed viability and ageing. This study investigated the relationship between seed viability and the individual half-cell reduction potentials (E_is) of four low-molecular-weight (LMW) thiols in Lathyrus pratensis seeds subjected to artificial ageing: GSH, cysteine (Cys), cysteinyl-glycine (Cys-Gly) and γ-glutamyl-cysteine (γ-Glu-Cys). The standard redox potential of γ-Glu-Cys was previously unknown and was experimentally determined. The E_is were mathematically combined to define a LMW thiol-disulphide based redox environment (E_{thiol-disulphide}). Loss of seed viability correlated with a shift in E_{thiol-disulphide} towards more positive values, with a LD₅₀ value of ?0.90 ± 0.093 mV M (mean ± SD). The mathematical definition of E_{thiol-disulphide} is envisaged as a step towards the definition of the overall cellular redox environment, which will need to include all known redox-couples.     

Occurrence and some properties of a novel γ-glutamyltransferase responsible for the syntheis of γ-L-glutamul-D-alanine in pea seedlings

Occurrence of a novel γ-glutamyltransferase responsible for the formation of γ-L-glutamyl-D-alanine was demonstrated in pea seedlings, and the enzyme was purified 600-fold. The enzyme preparation catalyzed the transfer of the γ-glutamyl moiety of L-glutamine and other γ-glutamyl compounds to D-amino acids. In the formation of γ-L-glutamyl peptides of D-amino acids, L-glutamine served as the most effective γ-glutamyl donor and D-alanine acted as a highly-specific acceptor. The maximum activity of the γ-glutamyl transfer reaction between L-glutamine and D-alanine was observed at pH 9.5 and the apparent K_m values for these amino acids were estimated to be 2.0 and 2.9mM, respectively. This unique γ-glutamyltransferase activity was always accompanied by the catalytic activities of the known γ-glutamyltransferases during the purification procedure.     

Isolation and characterization of key contributors to the “kokumi” taste in soybean seeds

The water extract of soybean seeds (Glycine max (L.) Merr.) is nearly tasteless, but “kokumi” taste sensation was confirmed upon addition of a basic umami solution containing glutamic acid, inosine monophosphate, and sodium chloride. To identify the key contributors to the “kokumi” taste sensation in soybean seeds, sensory-guided fractionation, taste sensory analyses, and LC–MS/MS analyses were utilized. γ-glutamyl-tyrosine and γ-glutamyl-phenylalanine were identified as contributors to “kokumi taste”; specifically, these γ-glutamyl peptides imparted the “kokumi” taste sensation at a low taste threshold in a basic umami solution. Raffinose and stachyose, which are sufficiently present in soybean seeds, exhibited a synergistic effect in regard to the enhanced “kokumi” taste sensation of γ-glutamyl peptides. This is the first report that the combined use of γ-glutamyl peptides and oligosaccharides can increase the “kokumi” intensity, which suggests that soybean extracts or soymilk can be used to enhance the “kokumi” taste sensation in food products.