-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694 (PnGGT) exhibited higher hydrolytic activity than transfer activity, as compared with other -glutamyltranspeptidases (GGTs). PnGGT showed little activity towards most of L-amino acids and towards glycyl-glycine, which is often used as a standard -glutamyl accepter in GGT transfer reactions. The preferred substrates for PnGGT as a -glutamyl accepter were amines such as methylamine, ethylamine, and isopropylamine.

Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ϒ-glutamyltranspeptidase (GGT) from Pseudomonas nitroreducens IFO12694 (PnGGT) has a unique preference for primary amines as ϒ-glutamyl acceptors over standard L-amino acids and peptides. This characteristic is useful for the synthesis of theanine. We used X-ray crystallographic analysis to understand the structural basis of PnGGT’s hydrolysis and transpeptidation reactions and to characterize its previously unidentified acceptor site. Structural studies of PnGGT have shown that key interactions between three residues (Trp385, Phe417, and Trp525) distinguish PnGGT from other GGTs. We studied the roles of these residues in the distinct biochemical properties of PnGGT using site-directed mutagenesis. All mutants showed a significant decrease in hydrolysis activity and an increase in transpeptidase activity, suggesting that the aromatic side chains of Trp385, Phe417, and Trp525 were involved in the recognition of acceptor substrates. Abbreviations: ϒ-glutamyl peptide, theanine, X-ray crystallography.

Theanine (γ-glutamylethylamide) is the major "umami" component in tea and exhibits various favorable physiological effects in mammals. To quantify theanine contained in commercial tea beverages, a coupled reaction of γ-glutamyltranspeptidase from Pseudomonas nitroreducens NBRC 12694 and amine dehydrogenase from Paracoccus denitrificans NBRC 12442 was investigated. This method measures changes in the absorbance of 2, 6-dichloroindophenol at 600 nm and is fast (less than 10 min, including the time required for sample pretreatment). The calibration curve was linear between theanine concentrations of 0 − 200 μM, with R 2 = 0.998. Theanine concentrations in samples of commercial tea beverages (black tea, green tea, and oolong tea) obtained using this method were similar to those obtained using the pre-column derivatization HPLC method. The proposed colorimetric assay for theanine determination is a reliable and timesaving method and can be used in food quality control and biological research.