Hiroki Ikezoe
Ritsumeikan University, Japan
Title: Function of the tryptophan residure in γ-glutamyltranspeptidase from Pseudomonas nitroreducens
Biography
Biography: Hiroki Ikezoe
Abstract
γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the transfer of the γ-glutamyl moiety from γ-glutamyl compounds to acceptor substrates or the hydrolysis of γ-glutamyl compounds. GGT from Pseudomonas nitroreducens (PnGGT) is used for the industrial synthesis of γ-glutamyl-ethylamide (theanine), which contributes the fifth taste that is known as umami. However, the detailed properties of PnGGT are still unclear. Hence, it is necessary to reveal the structural basis which is involved in hydrolysis and transpeptidation reactions and to identify the acceptor site of PnGGT for more efficient synthesis of theanine. Structural studies of PnGGT have shown that the three amino acid residues (Trp385, Phe417, and Trp525) might be involved in the recognition of acceptor substrates. Here we further investigated the role of Trp385 in PnGGT through site-directed mutagenesis and biochemical analyses.
We substituted Trp385 by the other four amino acids (Ala, Ser, Asp and Lys) and got PnGGT mutants (W385A, W385S, W385D and W385K). W385A was expressed as a precursor form in insoluble fraction, and W385D was not expressed in any fractions, whereas W385S and W385K were expressed as a mature form in soluble fraction. The activity assays with p-nitroaniline or γ-l-glutamyl hydroxamate showed that substitution of Trp385 resulted in drastic decrease in hydrolysis activity and significant increase in transpeptidase activity. W385S and W385K had a lower substrate specificity towards ethylamine compared to wild-type PnGGT. These results suggested that Trp385 was essential residue for auto processing, catalytic reaction and substrate recognition mechanism of PnGGT.