γ-Glutamyltranspeptidase essential for the metabolism of γ-glutamyl compounds in bacteria and its application.

Abstract

The enzymatic characteristics of γ-glutamyltranspeptidase were elucidated. The catalytic nucleophile of the enzymatic reaction of Escherichia coli γ-glutamyltranspeptidase was identified as the Oγ of the N-terminal Thr-residue of the small subunit. It was demonstrated that the inactive precursor of γ-glutamyltranspeptidase is processed autocatalytically and intramolecularly into the active heterodimeric mature enzyme via an ester intermediate. The catalytic nucleophile of this processing reaction was identified as the same Oγ atom of the N-terminal Thr-residue of the small subunit. These results were also supported by the three-dimensional structures of the γ-glutamyl enzyme intermediate and of the precursor-mimicked T391A nonprocessable mutant enzyme. Applications of transpeptidation and hydrolysis activities of bacterial γ-glutamyltranspeptidases were developed. Using transpeptidation activity, efficient enzymatic production of useful γ-glutamyl compounds, such as prodrug for Parkinson's disease, theanine and kokumi compound, was enabled. Hydrolysis activity was used as glutaminase and the mutant enzymes gaining glutaryl-7-aminocephalosporanic acid acylase activity were isolated.