Functional characterization of the extra sequence in the large subunit of γ-glutamyl transpeptidase from Bacillus atrophaeus: Role in autoprocessing and activity

Abstract

Abstract Bacterial γ-glutamyl transpeptidases (GGTs) exhibit differences in their catalytic properties due to distinct structural variability. Comparative sequence alignment of some well-characterized bacterial GGTs indicated marked differences at C-terminus in the large subunit of enzyme. An extra sequence of 14 amino acids was identified from genus Bacillus that was distinctly absent in other GGTs. Detailed analysis revealed five variable positions (V1 to V5) within this extra sequence encompassing four conserved positions and a consensus sequence XXX V X QP X DK XX G X (X indicates variable residue) was deduced. V1-V5 positions in GGT from Bacillus atrophaeus (BaGGT) were rationally mutated to understand their role in autoprocessing and catalysis. Seven substitutions at position V1 (E374) indicated its functional role in autoprocessing and catalytic activity further supported by in silico analysis showing vital interaction between residues 374 and 547 in creating proper autocatalytic environment. While thirteen mutations towards mapping role of positions V2-V5 suggested their involvement in proper in vivo folding and substrate accessibility within the binding pocket. Among all constructs, four variants with improved catalytic activity than native BaGGT were characterized and compared. One engineered variant E374 N at V1 position showed efficient autoprocessing, highest thermal stability (t1/2 of 64 min at 50 °C) and better transpeptidase activity (1.8 fold).