Expression and characterization of C-terminal truncated mutants of γ-glutamyltranspeptidase II (PaGGTII) from Pseudomonas aeruginosa PAO1

Abstract

The gene encoding γ-glutamyltranspeptidase II (PaGGTII) from Pseudomonas aeruginosa PAO1 was cloned in Escherichia coli. Recombinant PaGGTII showed a weak activity (0.0332 U/mg), and it can be easily inactivated. Multiple alignment of microbial GGTs showed the redundancy of the C-terminal of the small subunit of PaGGTII in length. The truncation of eight amino acid residues at the C-terminal of PaGGTII remarkably improved the activity and stability of the enzyme (PaGGTIIΔ8; 0.388 U/mg). Further truncation at the C-terminal also provided the enzyme relatively higher activity (PaGGTIIΔ9, -Δ10, -Δ11, and -Δ12). Among C-terminal truncated mutants, we focused on PaGGTIIΔ8 and examined the effect of C-terminal amino acid residues on the properties of PaGGTIIΔ8 because the activity of PaGGTII was found to be greatly improved when 8 amino acid residues were truncated. Various mutant enzymes with different C-terminal amino acid residues were constructed. They were expressed in E. coli and purified to homogeneity by ion-exchange chromatography. The properties of PaGGTIIΔ8 and the mutants obtained from mutation at E569 were characterized. K_m and k_cat of PaGGTIIΔ8 for γ-glutamyl-p-nitroanilide (γ-GpNA) were 8.05 mM and 15.49 s^-1, respectively. PaGGTIIΔ8E569Y showed the highest catalytic efficiency for γ-GpNA with a k_cat/Km of 12.55 mM^-1 s^-1. Mg²⁺, Ca²⁺, and Mn²⁺ exhibited positive effects on the catalytic activity for PaGGTIIΔ8 and its ten E569 mutants.

Keywords: C-terminal; Characterization; Pseudomonas aeruginosa PAO1; Truncated mutants; γ-glutamyltranspeptidase.