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

• Published in: Enzyme and microbial technology 2018-07, Vol.114, p.55-62
• Main Authors: Calvio, Cinzia, Romagnuolo, Fabio, Vulcano, Francesca, Speranza, Giovanna, Morelli, Carlo F.
• Format: Article
• Language: English
• Subjects: Hydrolysis reaction; Lid-loop; Poly-γ-glutamic acid; Transpeptidation reaction; γ-glutamyltranspeptidase
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2018.04.001

[Display omitted] •A mutant GGT was obtained by inserting the lid loop of E. coli GGT into the structure of B. subtilis GGT.•The role of the lid loop in GGTs was investigated by comparing the activities of the mutant and wt enzymes.•The lid loop regulates the access of substrates into the active site, depending on molecular size.•The presence of the lid loop enhances the transpeptidase activity of the mutant enzyme with respect to the wt counterparts. 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.