Autoprocessing of Helicobacter pylori γ-Glutamyltranspeptidase Leads to the Formation of a Threonine-Threonine Catalytic Dyad

Abstract

Helicobacter pylorigamma-glutamyltranspeptidase (HpGT) is a glutathione-degrading enzyme that has been shown to be a virulence factor in infection. It is expressed as a 60-kDa inactive precursor that must undergo autocatalytic processing to generate a 40-kDa/20-kDa heterodimer with full gamma-glutamyl amide bond hydrolase activity. The new N terminus of the processed enzyme, Thr-380, is the catalytic nucleophile in both the autoprocessing and enzymatic reactions, indicating that HpGT is a member of the N-terminal nucleophile hydrolase superfamily. To further investigate activation as a result of autoprocessing, the structure of HpGT has been determined to a resolution of 1.9 A. The refined model contains two 40-kDa/20-kDa heterodimers in the asymmetric unit and has structural features comparable with other N-terminal nucleophile hydrolases. Autoprocessing of HpGT leads to a large conformational change, with the loop preceding the catalytic Thr-380 moving >35 A, thus relieving steric constraints that likely limit substrate binding. In addition, cleavage of the proenzyme results in the formation of a threonine-threonine dyad comprised of Thr-380 and a second conserved threonine residue, Thr-398. The hydroxyl group of Thr-398 is located equidistant from the alpha-amino group and hydroxyl side chain of Thr-380. Mutation of Thr-398 to an alanine results in an enzyme that is fully capable of autoprocessing but is devoid of enzymatic activity. Substrate docking studies in combination with homology modeling studies of the human homologue reveal additional mechanistic details of enzyme maturation and activation, substrate recognition, and catalysis.

Human γ-Glutamyl Transpeptidase 1

“…1) (13, 14). Affinity labeling and structural studies of bacterial GGTs revealed that the threonine at the N terminus of the small subunit acts as the catalytic nucleophile in Escherichia coli and Helicobacter pylori GGT (15,16). Using mass spectrometry analysis of inhibitor-bound hGGT1, Castonguay et al (17) identified Thr-381 as the catalytic nucleophile in the human enzyme.…”

Molecular Cloning and Characterization of γ-Glutamyltranspeptidase fromPseudomonas nitroreducensIFO12694

“…It is thought to play key roles in glutathione metabolism in both prokaryotic and eukaryotic organisms. Since its discovery in the sheep kidney, 1) it has been isolated and characterized from various sources such as humans, 2) rats, 3) radishes, 4) fungi, 5) Escherichia coli, 6) Helicobacter pylori, 7) and Bacillus subtilis.…”

Biochemical and structural characterization ofKlebsiella pneumoniaeoxamate amidohydrolase in the uric acid degradation pathway

“…However, T398S H. pylori GGT was still able to autoprocess into and subunits, although the t 1/2 for processing was increased 1.4-fold. In contrast, T342A HpxW did not autoprocess (data not shown; Boanca et al, 2007).…”

“…GGT is a relatively recent addition to the Ntn-hydrolase superfamily (Suzuki & Kumagai, 2002). Many members of the Ntn-hydrolase superfamily have been structurally characterized, including B. subtilis glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (Smith et al, 1994), E. coli penicillin G acylases and the E. coli, H. pylori and B. subtilis GGTs (Boanca et al, 2007;Okada et al, 2006;Wada et al, 2010). BLAST results suggested that HpxW is a member of the GGT family, which has modest sequence identity among its members ($30%; Suzuki et al, 1989).…”

“…GGTs usually contain a catalytic dyad consisting of either threonine-serine or threonine-threonine (Boanca et al, 2007). The first member of the dyad is located on the N-terminus of the newly formed subunit and is essential for both the autoprocessing of the proenzyme and for the catalytic reactions.…”