gamma-Glutamyl cysteine ligases (Gcls) catalyze the first step of glutathione synthesis in prokaryotes and many eukaryotes. This study aimed to determine the biochemical properties of three different Gcls from strains of Limosilactobacillus reuteri that accumulate gamma-glutamyl dipeptides. Gcl1, Gcl2, and Gcl3 were heterologously expressed in Escherichia coli and purified by affinity chromatography. Gcl1, Gcl2, and Gcl2 exhibited biochemical with respect to the requirement for metal ions, the optimum pH and temperature of activity, and the kinetic constants for the substrates cysteine and glutamate. The substrate specificities of the three Gcls to 14 amino acids were assessed by liquid chromatography-mass spectrometry. All three Gcls converted ala, met, glu, and gln into the corresponding gamma-glutamyl dipeptides. None of the three were activewith val, asp, and his. Gcl1 and Gcl3 but not Gcl2 formed gamma-glu-leu, gamma-glu-ile, and gamma-glu-phe; Gcl3 exhibited stronger activity with gly, pro, and asp when compared to Gcl2. Phylogenetic analysis of Gcl and the Gcl-domain of GshAB in lactobacilli demonstrated that most of Gcls were present in heterofermentative lactobacilli, while GshAB was identified predominantly in homofermentative lactobacilli. This distribution suggests a different ecological role of the enzyme in homofermentative and heterofermentative lactobacilli. In conclusion, three Gcls exhibited similar biochemical properties but differed with respect to their substrate specificity and thus the synthesis of kokumi-active gamma-glutamyl dipeptides.