Elucidation of Broad Substrate Specificity of a Novel γ-Glutamyl Transferase from Bacillus atrophaeus and Rational...

γ-Glutamyl methionine (γ-Glu-Met) is a potential kokumi substance as a food ingredient with an exceptionally low taste threshold. This study proposes the γ-glutamyl transferase from Bacillus atrophaeus (BaGGT) as a superior enzyme that outperforms other candidates including previously reported enzymes. BaGGT displayed a superior specific activity (413 U/mg) for l-methionine (Met) under 20:1 molar ratio of Met to l-glutamine (Gln), highlighting its potentiality as a highly efficient catalyst for γ-Glu-Met production. Harnessing BaGGT's outstanding performance, 411 mM γ-Glu-Met production with 82% conversion was achieved under 500 mM Gln and 1 M Met surpassing all previous records for enzymatic γ-Glu-Met synthesis. Next, the structure of the BaGGT acceptor binding pocket was determined, leading to the development of the T205F/V414F mutant through rational design. This mutant demonstrated enhanced substrate selectivity and efficiency, producing 95 mM γ-Glu-Met with 95% conversion under excess acceptor conditions, a result unachievable by the wild type. Our comprehensive analysis of BaGGT also provided novel insights into the self-proteolytic cleavage mechanism and molecular characteristics of the acceptor binding pocket. This research establishes an efficient production platform for γ-Glu-Met while contributing to a fundamental understanding of γ-glutamyl transferases, paving the way for future enzyme engineering efforts in the field of flavor enhancement and food biotechnology.