Data for the synthesis of oligo-γ-glutamylglutamines as model compounds for γ-glutamyltransferases (GGTs) and for normalization of activities of different GGTs.

γ-Glutamyltransferases (GGTs) are widespread, conserved enzymes that catalyze the transfer of the γ-glutamyl moiety from a donor substrate to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a γ-glutamyl enzyme intermediate. Although the vast majority of the known GGTs has a short sequence called lid-loop covering the glutamate binding site, Bacillus subtilis GGT and some other enzymes from Bacillus spp. lack the lid loop. In order to assess the possible role of the lid loop of GGTs in substrate selection, synthetic oligo-γ-glutamylglutamines containing up to three γ-glutamyl residues were used as model substrates. The activities of the enzymes under investigation were standardized with respect to a common reaction to ensure comparable results. The activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from Escherichia coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT (Calvio et al., 2018) [1]. Here we report the experimental procedures for the synthesis of model substrates γ-glutamylglutamines through the method of the N-phtaloyl-L-glutamic acid anhydride and the spectral data of the synthetized compounds. The data obtained in the normalization procedure of the activities of the three enzymes are also reported.

Evidences on the role of the lid loop of γ-glutamyltransferases (GGT) in substrate selection.

γ-Glutamyltransferase (GGT) catalyzes the transfer of the γ-glutamyl moiety from a donor substrate such as glutathione to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a γ-glutamyl enzyme intermediate. The vast majority of the known GGTs has a short sequence covering the glutamate binding site, called lid-loop. Although being conserved enzymes, both B. subtilis GGT and the related enzyme CapD from B. anthracis lack the lid loop and, differently from other GGTs, both accept poly-γ-glutamic acid (γ-PGA) as a substrate. Starting from this observation, in this work the activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from E. coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT. Results indicate that the absence of the lid loop seems not to be the sole structural feature responsible for the recognition of a polymeric substrate by GGTs. Nevertheless, time course of hydrolysis reactions carried out using oligo-γ-glutamyl glutamines as substrates showed that the lid loop acts as a gating structure, allowing the preferential selection of the small glutamine with respect to the oligomeric substrates. In this respect, the mutant B. subtilis GGT revealed to be more similar to E. coli GGT than to its wild-type counterpart. In addition, the transpeptidase activity of the newly produced mutant enzyme revealed to be higher with respect to that of both E. coli and wild-type B. subtilis GGT. These findings can be helpful in selecting GGTs intended as biocatalysts for preparative purposes as well as in designing mutant enzymes with improved transpeptidase activity.