title: Corynebacterium glutamicum ggtB encodes a functional γ-glutamyl transpeptidase with γ-glutamyl dipeptide synthetic and hydrolytic activity.
abstract: In this work the role of γ-glutamyl transpeptidase in the metabolism of γ-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 γ-glutamyl-p-nitroanilide. Highest activity of the enzyme was measured in sodium bicarbonate buffer at pH 9.6 and 45°C. The KM value was 123μM. GgtB catalyzed the concentration-dependent synthesis and hydrolysis of γ-glutamyl dipeptides and showed strong glutaminase activity. The intracellular concentrations of five γ-glutamyl dipeptides (γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Val, γ-Glu-Leu, γ-Glu-Met) were determined by HPLC-MS and ranged from 0.15 to 0.4mg/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.
authors.affiliation: Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Walter
authors.affiliation: Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Grenz
authors.affiliation: Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Ortseifen
authors.affiliation: Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Persicke
authors.affiliation: Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany. Electronic address: Joern.Kalinowski@Cebitec.Uni-Bielefeld.DE.
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D004151:Dipeptides / Q000032:analysis / Q000378:metabolism*; D006868:Hydrolysis; D005723:gamma-Glutamyltransferase / Q000235:genetics* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D004151:Dipeptides; D005723:gamma-Glutamyltransferase
keywords: Corynebacterium glutamicum; Gamma-glutamyl transpeptidase; Kokumi peptides; LC–MS; Metabolic profiling