PUB - Publikationen an der Universität Bielefeld

In this work the role of gamma-glutamyl transpeptidase in the metabolism of gamma-glutamyl dipeptides produced by Corynebacterium glutamicum ATCC 13032 was studied. The enzyme is encoded by the gene ggtB (cg1090) and synthesized as a 657 amino acids long preprotein. Gamma-glutamyl transpeptidase activity was found to be associated with intact cells of C. glutamicum and was abolished upon deletion of ggtB. Bioinformatic analysis indicated that the enzyme is a lipoprotein and is attached to the outer side of the cytoplasmic membrane. Biochemical parameters of recombinant GgtB were determined using the chromogenic substrate gamma-glutamyl-p-nitroanilide. Highest activity of the enzyme was measured in sodium bicarbonate buffer at pH 9.6 and 45 degrees C. The K-M value was 123 mu M. GgtB catalyzed the concentration-dependent synthesis and hydrolysis of gamma-glutamyl dipeptides and showed strong glutaminase activity. The intracellular concentrations of five gamma-glutamyl dipeptides (gamma-Glu-Glu,gamma-Glu-Glu,gamma-Glu-Val, gamma-Glu-Met) were determined by HPLC-MS and ranged from 0.15 to 0.4 mg/g CDW after exponential growth in minimal media. Although deletion and overexpression of ggtB had significant effects on intracellular dipeptide concentrations, it was neither essential for biosynthesis nor catabolism of these dipeptides in vivo. (C) 2015 Elsevier B.V. All rights reserved.