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

Abstract

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.

Expression of Bacillus amyloliquefaciens γ-Glutamyltransferase in Lactococcus lactis and Immobilization on Magnetic Nanoparticles

“…In this study, a phylogenetic analysis indicated that the gene of BaGGT and Bacillus GGT genes belonged to the same branch, on which basis we speculated that the primary structure of BaGGT was similar to that of known Bacillus GGTs . We aimed at providing a suitable expression host for this enzyme; a recombinant expression system for BaGGT in L.…”

“…In this study, a phylogenetic analysis indicated that the gene of BaGGT and Bacillus GGT genes belonged to the same branch, on which basis we speculated that the primary structure of BaGGT was similar to that of known Bacillus GGTs. 35 We aimed at providing a suitable expression host for this enzyme; a recombinant expression system for BaGGT in L. lactis (pNZ8048-sp usp45 -ggt Δsp -6his) was constructed. In addition, L. lactis is a well-known heterologous host with several advantages such as a reduced number of extracellular secreted proteins and proteases, which simplifies production and purification or the protein of interest and is an available toolbox for molecular manipulation.…”

Enzymatic Synthesis of γ-Glutamylvalylglycine Using Bacterial γ-Glutamyltranspeptidase

“…18 Many amino acids, peptides, and amines can be γ-glutamyl acceptors of bacterial GGT to synthesize γ-glutamyl compounds. 19,20 For example, enzymatic methods for synthesizing L-theanine (γglutamylethylamide), 21−23 γ-D-Glu-L-Trp, 24 γ-Glu-Val, 18,25 γ-Glu-Phe, 26 and γ-Glu-Gln 27 have been reported. We previously reported that valylglycine (Val-Gly) was the best γ-glutamyl acceptor for E. coli GGT among dipeptides whose C-terminal side was glycine; 28 therefore, E. coli GGT is considered to be a suitable enzyme for synthesizing γ-Glu-Val-Gly when Val-Gly is used for the substrate.…”

“…As the transpeptidation reaction of Escherichia coli GGT was activated by several cations at alkaline pH, the production yield of γ-Glu-Val increased approximately 1.5 times by the addition of 50 mM MgCl 2 to the reaction mixture after 3 h of incubation . Many amino acids, peptides, and amines can be γ-glutamyl acceptors of bacterial GGT to synthesize γ-glutamyl compounds. , For example, enzymatic methods for synthesizing l -theanine (γ-glutamylethylamide), − γ- d -Glu- l -Trp, γ-Glu-Val, , γ-Glu-Phe, and γ-Glu-Gln have been reported. We previously reported that valylglycine (Val-Gly) was the best γ-glutamyl acceptor for E.…”

Utilization of L-glutamate as a preferred or sole nutrient in Pseudomonas aeruginosa PAO1 depends on genes encoding for the enhancer-binding protein AauR, the sigma factor RpoN and the transporter complex AatJQMP

“…The enzyme Ggt catalyzes the transfer of γ-glutamyl groups from donor molecules, most notably glutathione, to target or acceptor substrates consisting of amino acids, peptides or even water. Although Ggt has been implicated in the metabolism of glutathione and cysteine in some bacteria [ 36 ], little is known on the biological importance of this enzyme in Pseudomonas , including any potential role it may have in glutamate-related metabolism.

Fig.

…”