Bacterial Γ-glutamyltranspeptidase Research Articles

Enzymatic Synthesis of γ-Glutamylvalylglycine Using Bacterial γ-Glutamyltranspeptidase.

γ-Glutamylvalylglycine is known as a potent "kokumi" substance that increases the thickness, continuity, and mouthfulness of the taste of food. In this study, γ-glutamylvalylglycine was enzymatically synthesized from glutamine and valylglycine through the transpeptidation reaction of bacterial γ-glutamyltranspeptidase. The reaction conditions decided for the production of γ-glutamylvalylglycine are 20 mM glutamine, 100 mM valylglycine, 30 mU/mL γ-glutamyltranspeptidase, and 0.856 M (5%) NaCl at pH 8 and 37 °C, and 18.6 mM γ-glutamylvalylglycine was synthesized. Metal cations, Na+ and Mg2+, improved the transpeptidation reaction of γ-glutamyltranspeptidase, and the production of γ-glutamylvalylglycine increased. Moreover, by feeding a suitable amount of glutamine every 5 h, the maximum production of γ-glutamylvalylglycine increased 1.7 times after 20 h of incubation.

Functional characterization of the extra sequence in the large subunit of γ-glutamyl transpeptidase from Bacillus atrophaeus: Role in autoprocessing and activity

Bacterial γ-glutamyl transpeptidases (GGTs) exhibit differences in their catalytic properties due to distinct structural variability. Comparative sequence alignment of some well-characterized bacterial GGTs indicated marked differences at C-terminus in the large subunit of enzyme. An extra sequence of 14 amino acids was identified from genus Bacillus that was distinctly absent in other GGTs. Detailed analysis revealed five variable positions (V1 to V5) within this extra sequence encompassing four conserved positions and a consensus sequence XXXVXQPXDKXXGX (X indicates variable residue) was deduced. V1-V5 positions in GGT from Bacillus atrophaeus (BaGGT) were rationally mutated to understand their role in autoprocessing and catalysis. Seven substitutions at position V1 (E374) indicated its functional role in autoprocessing and catalytic activity further supported by in silico analysis showing vital interaction between residues 374 and 547 in creating proper autocatalytic environment. While thirteen mutations towards mapping role of positions V2-V5 suggested their involvement in proper in vivo folding and substrate accessibility within the binding pocket. Among all constructs, four variants with improved catalytic activity than native BaGGT were characterized and compared. One engineered variant E374 N at V1 position showed efficient autoprocessing, highest thermal stability (t1/2 of 64 min at 50 °C) and better transpeptidase activity (1.8 fold).

γ-Glutamyltranspeptidase essential for the metabolism of γ-glutamyl compounds in bacteria and its application.

The enzymatic characteristics of γ-glutamyltranspeptidase were elucidated. The catalytic nucleophile of the enzymatic reaction of Escherichia coli γ-glutamyltranspeptidase was identified as the Oγ of the N-terminal Thr-residue of the small subunit. It was demonstrated that the inactive precursor of γ-glutamyltranspeptidase is processed autocatalytically and intramolecularly into the active heterodimeric mature enzyme via an ester intermediate. The catalytic nucleophile of this processing reaction was identified as the same Oγ atom of the N-terminal Thr-residue of the small subunit. These results were also supported by the three-dimensional structures of the γ-glutamyl enzyme intermediate and of the precursor-mimicked T391A nonprocessable mutant enzyme. Applications of transpeptidation and hydrolysis activities of bacterial γ-glutamyltranspeptidases were developed. Using transpeptidation activity, efficient enzymatic production of useful γ-glutamyl compounds, such as prodrug for Parkinson's disease, theanine and kokumi compound, was enabled. Hydrolysis activity was used as glutaminase and the mutant enzymes gaining glutaryl-7-aminocephalosporanic acid acylase activity were isolated.

Bacterial γ-glutamyltranspeptidases, physiological function, structure, catalytic mechanism and application.

γ-Glutamyltranspeptidase (GGT) has been widely used as a marker enzyme of hepatic and biliary diseases and relations between various diseases and its activity have been studied extensively. Nevertheless, several of its fundamental enzymatic characteristics had not been elucidated. We obtained homogeneous preparation of GGTs from bacteria, characterized them, and elucidated its physiological function that is common to mammalian cells, using GGT-deficient E. coli. Prior to GGT of all living organisms, we also identified catalytic nucleophile of E. coli GGT and revealed the post-translational processing mechanism for its maturation, and also its crystal structure was determined. The reaction intermediate was trapped and the structure-based reaction mechanism was presented. As for its application, using its transferase activity, we developed the enzymatic synthesis of various γ-glutamyl compounds that are promising in food, nutraceutical and medicinal industries. We found GGT of Bacillus subtilis is salt-tolerant and can be used as a glutaminase, which is important in food industry, to enhance umami of food, such as soy sauce and miso. We succeeded in converting bacterial GGT to glutaryl-7-aminocephalosporanic acid acylase, which is an important enzyme in cephem antibiotics production, by site-directed and random mutagenesis.

Bacterial γ-glutamyltranspeptidase: Food and medicinal applications

Bacterial γ-glutamyltranspeptidase: Food and medicinal applications

Isolation of γ-Glutamyl-Transferase Rich-Bacteria from Mouse Gut by a Near-Infrared Fluorescent Probe with Large Stokes Shift.

Bacterial γ-glutamyltranspeptidases (γ-GT) is a well-known metabolic enzyme, which could cleave the γ-glutamyl amide bond of γ-glutamyl analogues. As a key metabolic enzyme of bacteria and a virulence factor for the host, bacterial γ-GT was determined to be a novel pharmaceutical target for new antibiotics development. However, there is no efficient method for the sensing of γ-GT activity in bacteria and the recognition of γ-glutamyltransferase rich-bacteria. In the present work, a dicyanoisophorone derivative (ADMG) has been designed and developed to be a sensitive and selective near-infrared fluorescent probe for the sensing of bacterial γ-GT. ADMG not only sensed bacterial γ-GT in vitro, but also imaged intestinal bacteria in vivo. More interesting, the intestinal bacteria existed in the duodenum section of mouse displayed significant fluorescence emission. Under the guidance of the sensing of γ-GT using ADMG, three intestinal bacteria strains K. pneumoniae CAV1042, K. pneumoniae XJRML-1, and E. faecalis were isolated successfully, which expressed the bacterial γ-GT. Therefore, the fluorescent probe ADMG not only sensed the endogenous bacterial γ-GT and imaged the intestinal bacteria but also guided the isolation of intestinal bacteria possessing γ-GT efficiently, which suggested a novel biological tool for the rapid isolation of special bacteria from a mixed sample.

Role of bacterial γ-glutamyltranspeptidase as a novel virulence factor in bone-resorbing pathogenesis.

Mammalian γ-glutamyltranspeptidase (GGT) has been identified as a bone-resorbing factor. Since GGT of Bacillus subtilis exhibits similarity in their primary structure and enzymatic characteristics with mammalian GGTs, the bone-resorbing activity of bacterial GGT was examined in this study. Osteoclastogenesis was performed in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. A conditioned medium from GGT-overproducing B. subtilis culture showed significantly higher activity of osteoclast formation than a conditioned medium from wild-type B. subtilis culture. Recombinant GGT (rGGT) of wild-type B. subtilis and an enzymatic activity-defected rGGT of B. subtilis 2288 mutant were expressed in Escherichia coli and purified using His tag. Both purified rGGTs induced similar levels of osteoclastogenesis, suggesting that B. subtilis GGT possesses virulent bone-resorbing activity and its activity is probably independent of its enzymatic activity. Furthermore, a recombinant protein of B. subtilis GGT heavy subunit (Bs rGGT/H) showed strong activity of osteoclastogenesis while the light subunit failed to show strong activity, suggesting that the bone-resorbing activity is mainly located at the heavy subunit. More importantly, the GGT enzymatic activity may not be required for this virulence activity since the light subunit contains the catalytic pocket. In addition, B. subtilis rGGT stimulated mRNA expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and cyclooxygenase-2 (COX-2), while an osteoprotegerin inhibited the osteoclast formation induced by Bs rGGT/H. This is the first demonstration that bacterial GGT itself is sufficient to act as a bone-resorbing virulence factor via RANKL-dependent pathway. Therefore, it can be hypothesized that GGT of periodontopathic bacteria may play an important role as a virulence factor in bone destruction.

Surface-functionalized hyperbranched poly(amido acid) magnetic nanocarriers for covalent immobilization of a bacterial γ-glutamyltranspeptidase.

In this study, we synthesized water-soluble hyperbranched poly(amido acid)s (HBPAAs) featuring multiple terminal CO2H units and internal tertiary amino and amido moieties and then used them in conjunction with an in situ Fe2+/Fe3+ co-precipitation process to prepare organic/magnetic nanocarriers comprising uniformly small magnetic iron oxide nanoparticles (NP) incorporated within the globular HBPAAs. Transmission electron microscopy revealed that the HBPAA-γ-Fe2O3 NPs had dimensions of 6–11 nm, significantly smaller than those of the pristine γ-Fe2O3 (20–30 nm). Subsequently, we covalently immobilized a bacterial γ-glutamyltranspeptidase (BlGGT) upon the HBPAA-γ-Fe2O3 nanocarriers through the formation of amide linkages in the presence of a coupling agent. Magnetization curves of the HBPAA-γ-Fe2O3/BlGGT composites measured at 300 K suggested superparamagnetic characteristics, with a saturation magnetization of 52 emu g−1. The loading capacity of BlGGT on the HBPAA-γ-Fe2O3 nanocarriers was 16 mg g−1 support; this sample provided a 48% recovery of the initial activity. The immobilized enzyme could be recycled 10 times with 32% retention of the initial activity; it had stability comparable with that of the free enzyme during a storage period of 63 days. The covalent immobilization and stability of the enzyme and the magnetization provided by the HBPAA-γ-Fe2O3 NPs suggests that this approach could be an economical means of depositing bioactive enzymes upon nanocarriers for BlGGT-mediated bio-catalysis.

Increase of “Umami” and “Kokumi” Compounds in Miso, Fermented Soybeans, by the Addition of Bacterial γ-Glutamyltranspeptidase

γ-Glutamyltranspeptidase (GGT) hydrolyzes γ-glutamyl compounds and transfers their γ-glutamyl moieties to amino acids and peptides. We previously showed that the “umami” taste of soy sauce could be improved by the addition of salt-tolerant Bacillus subtilis GGT to the fermentation mixture, “moromi”. Although miso fermentation is a semi-solid fermentation, unlike soy sauce fermentation, this was also the case. When 15 units of purified B. subtilis GGT were added to 418 g miso “moromi” (fermentation mixture), the glutamate concentration in “moromi” became 20 mM higher and the “umami” taste became stronger than without the addition of GGT after 2 to 6 months of fermentation. In addition, γ-Glu-Val and γ-Glu-Val-Gly, which are known as “kokumi” peptides, were identified in “tamari”, and the concentrations of these γ-glutamyl peptides in “tamari" fermented by the addition of GGT were significantly higher than those of “moromi” without the addition of GGT. These results indicate that B. subtilis GGT is able to improve the taste of miso.

Increase of “Umami” and “Kokumi” Compounds in Miso, Fermented Soybeans, by the Addition of Bacterial γ-Glutamyltranspeptidase

γ-Glutamyltranspeptidase (GGT) hydrolyzes γ-glutamyl compounds and transfers their γ-glutamyl moieties to amino acids and peptides. We previously showed that the “umami” taste of soy sauce could be improved by the addition of salt-tolerant Bacillus subtilis GGT to the fermentation mixture, “moromi”. Although miso fermentation is a semi-solid fermentation, unlike soy sauce fermentation, this was also the case. When 15 units of purified B. subtilis GGT were added to 418 g miso “moromi” (fermentation mixture), the glutamate concentration in “moromi” became 20 mM higher and the “umami” taste became stronger than without the addition of GGT after 2 to 6 months of fermentation. In addition, γ-Glu-Val and γ-Glu-Val-Gly, which are known as “kokumi” peptides, were identified in “tamari”, and the concentrations of these γ-glutamyl peptides in “tamari" fermented by the addition of GGT were significantly higher than those of “moromi” without the addition of GGT. These results indicate that B. subtilis GGT is able to improve the taste of miso.

Improvement of the Glutaryl-7-Aminocephalosporanic Acid Acylase Activity of a Bacterial γ-Glutamyltranspeptidase

Improvement of the Glutaryl-7-Aminocephalosporanic Acid Acylase Activity of a Bacterial γ-Glutamyltranspeptidase

Improvement of the Glutaryl-7-Aminocephalosporanic Acid Acylase Activity of a Bacterial γ-Glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.

Improving the umami taste of soy sauce by the addition of bacterial γ-glutamyltranspeptidase as a glutaminase to the fermentation mixture

γ-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of γ-glutamyl compounds and transfer of their γ-glutamyl moieties to amino acids and peptides. GGT is able to catalyze the hydrolysis of glutamine (Gln) to glutamic acid (Glu), the so called glutaminase reaction. A salt-tolerant wild-type GGT from Bacillus subtilis and D445A mutant GGT, whose transpeptidation activity was abolished, were added to a soy sauce fermentation mixture to increase the amount of Glu in the product. The concentration of Glu in the soy sauce was 36 mM higher with the addition of wild-type GGT than without. Nine out of ten members of a panel recognized a difference and preferred the taste of the soy sauce fermented with the wild-type GGT. These results indicate that wild-type GGT is competent for use in the fermentation of soy sauce as a glutaminase.

γ-Glutamyl compounds and their enzymatic production using bacterial γ-glutamyltranspeptidase

Some amino acids and peptides, which have low solubility in water, become much more soluble following gamma-glutamylation. Compounds become more stable in the blood stream with gamma-glutamylation. Several gamma-glutamyl compounds are known to have favorable physiological effects on mammals. Gamma-glutamylation can improve taste and can stabilize glutamine in aqueous solution. Because of such favorable features, gamma-glutamyl compounds are very attractive. However, only a small number of gamma-glutamyl amino acids have been studied although many other gamma-glutamyl compounds may have characteristics that will benefit humans. This is mainly because gamma-glutamyl compounds have not been readily available. An efficient and simple method of producing various gamma-glutamyl compounds, especially gamma-glutamyl amino acids, using bacterial gamma-glutamyltranspeptidase has been developed. With this method, modifications of reactive groups of the substrate and energy source such as ATP are not required, and a wide-range of gamma-glutamyl compounds can be synthesized. Moreover, bacterial gamma-glutamyltranspeptidase, a catalyst for this method, is readily available from the strain over-producing this enzyme. The superiority of producing gamma-glutamyl compounds with bacterial gamma-glutamyltranspeptidase over other methods of production is discussed.

Development of an efficient enzymatic production of γ- d-glutamyl- l-tryptophan (SCV-07), a prospective medicine for tuberculosis, with bacterial γ-glutamyltranspeptidase

γ- d-Glutamyl- l-tryptophan (SCV-07) is a prospective medicine for the treatment of tuberculosis, according to the phase two clinical trial. Because γ- d-glutamyl- l-tryptophan has several reactive groups in its molecule, consists of d- and l-amino acids, and is connected by γ-glutamyl linkage, its chemical synthesis is complicated. An efficient enzymatic method to synthesize γ- d-glutamyl- l-tryptophan from d-glutamine and l-tryptophan employing bacterial γ-glutamyltranspeptidase was developed. The optimum reaction conditions were 50 mM d-glutamine, 50 mM l-tryptophan, and 0.2 U ml −1 γ-glutamyltranspeptidase, pH 9–9.5, and incubation at 37 °C for 5 h. After a 5 h incubation, 33 mM γ- d-glutamyl- l-tryptophan was obtained, the conversion rate being 66%. The product was purified by Dowex 1 × 8 column and was considered to be γ- d-glutamyl- l-tryptophan.

Use of bacterial gamma-glutamyltranspeptidase for enzymatic synthesis of gamma-D-glutamyl compounds.

An enzymatic method for synthesizing various gamma-D-glutamyl compounds efficiently and stereospecifically involving bacterial gamma-glutamyltranspeptidase (EC 2.3.2.2) with D-glutamine as a gamma-glutamyl donor was developed. With D-glutamine as a gamma-glutamyl donor instead of L-glutamine in gamma-glutamyltaurine synthesis, by-products such as gamma-glutamylglutamine and gamma-glutamyl-gamma-glutamyltaurine were not synthesized and the yield of gamma-glutamyltaurine dramatically increased from 25 to 71%. It was also shown that the purification could be simplified without these gamma-glutamyl by-products. The possibility of synthesizing various gamma-D-glutamyl compounds was also shown.

Enzymatic production of theanine, an “umami” component of tea, from glutamine and ethylamine with bacterial γ-glutamyltranspeptidase

Theanine (γ-glutamylethylamide) is the major “umami” (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of theanine involving bacterial γ-glutamyltranspeptidase (GGT) was developed. The optimum reaction conditions were 200 mM Gln, 1.5 M ethylamine, and 0.4 units/ml GGT, pH 10. After 2-h incubation at 37 °C, 120 mM theanine was obtained, the conversion rate against Gln being 60%. Theanine was purified on Dowex 50 W×8 and Dowex 1×8 columns, and then identified by 1 H -NMR. This is the first report that a GGT, regardless of its source, can utilize an alkyl amine as an acceptor of the γ-glutamyl moiety in its transpeptidation reaction.

Improvement of the bitter taste of amino acids through the transpeptidation reaction of bacterial gamma-glutamyltranspeptidase.

The tastes of several bitter amino acids and their gamma-glutamyl derivatives were compared. The bitterness of Phe, Val, Leu, and His was reduced, sourness was produced, and preferences were increased by gamma-glutamylization. Because the effect of gamma-glutamylization of bitter amino acids was most obvious for Phe, which is an atypical bitter amino acid, an enzymatic method for the synthesis of gamma-glutamylphenylalanine (gamma-Glu-Phe) involving bacterial gamma-glutamyltranspeptidase was developed. The optimum reaction conditions were 200 mM Gln, 200 mM Phe, and 0.5 unit/mL GGT, pH 10.4. After 1.5-h of incubation at 37 degrees C, 140 mM gamma-Glu-Phe was obtained, the yield being 70%. gamma-Glu-Phe was purified on a Dowex 1x8 column and then identified by NMR.