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Several γ-glutamyl compounds are known to have favorable features for a variety of biotechnological and pharmaceutical applications. However, so far only a limited number of γ-glutamyl compounds have been synthesized through γ-glutamyltranspeptidase (GGT). With the help of thin layer chromatography and computer-assisted image analysis techniques, we performed an enzymatic method involving Bacillus licheniformis GGT (BlGGT) for the synthesis of γ-glutamyl-L-leucine (γ-Glu- Leu). The optimum conditions for a biocatalytic synthesis of γ- Glu-Leu were determined to be 200 mM glutamine, 200 mM leucine, 50 mM Tris-HCl buffer (pH 9.0), and BlGGT at a working concentration of 2.0 U/mL. Upon a 5-h incubation of the reaction mixture at 50°C, the product yield could reach approximately 52%. The preparative synthesis of γ-Glu-Leu by BlGGT was done under optimal conditions at millimol scale level. The reaction products were subjected to mass spectrometry/nuclear magnetic resonance analyses to ensure the successful synthesis of γ-Glu-Leu. Taken together, the present study demonstrates the high potentiality of BlGGT-catalyzed reaction in the synthesis of γ-glutamyl compounds. [ABSTRACT FROM AUTHOR]

Several γ-glutamyl compounds are known to have attractive features for food industry applications. In this study, we report a straightforward procedure for the biocatalytic synthesis of γ-glutamyl-phenylalanine (γ-Glu-Phe) using recombinant Bacillus licheniformis γ-glutamyltranspeptidase ( Bl GGT). The qualitative analysis of reaction products was carried out by thin-layer chromatography technique and computer-assisted image analysis. The optimized conditions for the biocatalytic process were pH 9.0, 50 °C, l -glutamine and l -phenylalanine in a 1:1 ratio, and Bl GGT at a working concentration of 2.0 U/ml. Preparative synthesis of γ-Glu-Phe was also performed at mmol scale. The desired product from the reaction mixture was then verified by mass spectrometry/nuclear magnetic resonance analyses. Strikingly, the experimental results suggest the high potentiality of Bl GGT-catalyzed reaction in the enzymatic synthesis of γ-glutamyl peptides. [ABSTRACT FROM AUTHOR]

In the practical application of Bacillus licheniformis γ-glutamyltranspeptidase ( Bl GGT), 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 Bl GGT 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 Bl GGT. [ABSTRACT FROM AUTHOR]

Abstract: The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was explored by site-directed mutagenesis. This glutamate was substituted by either alanine, aspartate, arginine or glutamine and the expressed mutant enzymes were purified to apparent homogeneity with metal-affinity chromatography. SDS–PAGE analysis showed that E398A, E398D and E398K were unable to process themselves into a large and a small subunit. However, E398Q was not only able to process itself, but also had a catalytic activity comparable to that of BlGGT. As compared with the wild-type enzyme, no significant change in circular dichroism spectra was observed for the mutant proteins. Thermal unfolding of BlGGT, E398A, E398D, E398K and E398Q followed the two-state unfolding process with a transition point (T m) of 47.7–69.4 °C. Tryptophan fluorescence spectra of the mutant enzymes were different from the wild-type protein in terms of fluorescence intensity. Native BlGGT started to unfold beyond ∼1.92 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]0.5, N–U, at 3.07 M equivalent to free energy change () of 14.53 kcal/mol for the N → U process, whereas the denaturation midpoints for the mutant enzymes were 1.31–2.99 M equivalent to of 3.29–12.05 kcal/mol. Taken together, these results strongly suggest that the explored glutamate residue is indeed important for the autocatalytic processing of BlGGT. [Copyright &y& Elsevier]

A highly conserved 458PLSSMXP464 sequence in the small subunit (S-subunit) of an industrially important Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was identified by sequence alignment. Molecular structures of the precursor mimic and the mature form of BlGGT clearly reveal that this peptide sequence is in close spatial proximity to the self-processing and catalytic sites of the enzyme. To probe the role of this conserved sequence, ten mutant enzymes of BlGGT were created through a series of deletion and alanine-scanning mutagenesis. SDS-PAGE and densitometric analyses showed that the intrinsic ability of BlGGT to undergo autocatalytic processing was detrimentally affected by the deletion-associated mutations. However, loss of self-activating capacity was not obviously observed in most of the Ala-replacement mutants. The Ala-replacement mutants had a specific activity comparable to or greater than that of the wild-type enzyme; conversely, all deletion mutants completely lost their enzymatic activity. As compared with BlGGT, S460A and S461S showed greatly enhanced kcat/Km values by 2.73- and 2.67-fold, respectively. The intrinsic tryptophan fluorescence and circular dichroism spectral profiles of Ala-replacement and deletion mutants were typically similar to those of BlGGT. However, heat and guanidine hydrochloride-induced unfolding transitions of the deletion-associated mutant proteins were severely reduced as compared with the wild-type enzyme. The predictive mutant models suggest that the microenvironments required for both self-activation and catalytic reaction of BlGGT can be altered upon mutations. [ABSTRACT FROM AUTHOR]

γ-Glutamyl transpeptidases (γ-GTs) are members of N-terminal nucleophile hydrolase superfamily. They are synthetized as single-chain precursors, which are then cleaved to form mature enzymes. Basic aspects of autocatalytic processing of these pro-enzymes are still unknown. Here we describe the X-ray structure of the precursor mimic of Bacillus licheniformis γ-GT ( Bl GT), obtained by mutating catalytically important threonine to alanine (T399A- Bl GT), and report results of autoprocessing of mutants of His401, Thr415, Thr417, Glu419 and Arg571. Data suggest that Thr417 is in a competent position to activate the catalytic threonine (Thr399) for nucleophilic attack of the scissile peptide bond and that Thr415 plays a major role in assisting the process. On the basis of these new structural results, a possible mechanism of autoprocessing is proposed. This mechanism, which guesses the existence of a six-membered transition state involving one carbonyl and two hydroxyl groups, is in agreement with all the available experimental data collected on γ-GTs from different species and with our new Ala-scanning mutagenesis data. [ABSTRACT FROM AUTHOR]

Abstract: The oligomeric states of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases (BlGGT and EcGGT) in solution have been investigated by analytical ultracentrifugation. The results showed that BlGGT has a sedimentation coefficient of 5.12S, which can be transformed into an experimental molecular mass of approximately 62,680Da. The monomeric conformation is conserved in EcGGT. SDS-PAGE analysis and cross-linking studies further proved that the autocatalytically processed BlGGT and EcGGT form a heterodimeric association. Unfolding analyses using circular dichroism and tryptophan emission fluorescence revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and EcGGT had a T m value of 59.5 and 49.2°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Chemical unfolding of BlGGT was independent to the pH value ranging from 5 to 10, whereas the pH environment was found to significantly influence the GdnHCl-induced denaturation of EcGGT. Both enzymes did not reactivate from the completely unfolded states, accessible at 6M GdnHCl. BlGGT was active in the presence of 4M NaCl, whereas the activity of EcGGT was significantly decreased at the high-salt condition. Taken together, these findings suggest that the biophysical properties of the homologous GGTs from two mesophilic sources are quite different. [Copyright &y& Elsevier]