Crystal structure analysis and enzymatic characterization of -glutamyltranspeptidase from Pseudomonas nitroreducens

Theanine (-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ?-glutamyltranspeptidase (GGT) from Pseudomonas nitroreducens IFO12694 (PnGGT) has a unique preference for primary amines as ?-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: ?-glutamyl peptide, theanine, X-ray crystallography.