title: pH-dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ-glutamyltransferase.
abstract: γ-Glutamyltransferases (γ-GTs) are heterodimeric enzymes that catalyze the transfer of a γ-glutamyl group from a donor species to an acceptor molecule in a transpeptidation reaction through the formation of an intermediate γ-glutamyl enzyme. In our search for a γ-GT from a generally recognized as safe microorganism suitable for the production of γ-glutamyl derivatives with flavor-enhancing properties intended for human use, we cloned and overexpressed the γ-GT from Bacillus subtilis. In this study, we report the behavior of B. subtilis γ-GT in reactions involving glutamine as the donor compound and various acceptor amino acids. The common thread emerging from our results is a strong dependence of the hydrolase, transpeptidase and autotranspeptidase activities of B. subtilis γ-GT on pH, also in relation to the pKa of the acceptor amino acids. Glutamine, commonly referred to as a poor acceptor molecule, undergoes rapid autotranspeptidation at elevated pH, affording oligomeric species, in which up to four γ-glutamyl moieties are linked to a single glutamine. Moreover, we found that D-glutamine is also recognized both as a donor and as an acceptor substrate. Our results prove that the B. subtilis γ-GT-catalyzed transpeptidation reaction is feasible, and the observed activities of γ-GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material γ-polyglutamic acid.
authors.affiliation: Department of Chemistry, University of Milan, Italy; Italian Biocatalysis Center (IBC), c/o Dipartimento di Scienza del Farmaco, University of Pavia, Italy.
authors.forename: Carlo F
authors.initials: CF
authors.lastname: Morelli
authors.forename: Cinzia
authors.initials: C
authors.lastname: Calvio
authors.forename: Marco
authors.initials: M
authors.lastname: Biagiotti
authors.forename: Giovanna
authors.initials: G
authors.lastname: Speranza
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D005972:Glutaminase / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
chemical_list: D005973:Glutamine; D010458:Peptidyl Transferases; D005723:gamma-Glutamyltransferase; D006867:Hydrolases; D005972:Glutaminase
keywords: Bacillus subtilis; enzyme catalysis; poly-γ-glutamic acid; transpeptidation reaction; γ-glutamyltransferase