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

Abstract

Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure an improves immune responses. The ϒ-glutamyltranspeptidase (GGT) from Pseudomonas nitror educens IFO12694 (PnGGT) has a unique preference for primary amines as ϒ-glutamy acceptors over standard L-amino acids and peptides. This characteristic is useful for th synthesis of theanine. We used X-ray crystallographic analysis to understand the structura basis of PnGGT’s hydrolysis and transpeptidation reactions and to characterize its previousl unidentified acceptor site. Structural studies of PnGGT have shown that key interaction 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 usin site-directed mutagenesis. All mutants showed a significant decrease in hydrolysis activit and an increase in transpeptidase activity, suggesting that the aromatic side chains of Trp385 Phe417 and Tr 525 were involved in the reco nition of acce tor substrates.