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Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis ?-glutamyltranspeptidase.

ABSTRACT: The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis ?-glutamyltranspeptidase (BlGGT) was explored by site-directed mutagenesis. This glutamate was substituted by either alanine, aspartate, arginine or glutamine and the expressed mutant enzymes were purified to apparent homogeneity with metal-affinity chromatography. SDS-PAGE analysis showed that E398A, E398D and E398K were unable to process themselves into a large and a small subunit. However, E398Q was not only able to process itself, but also had a catalytic activity comparable to that of BlGGT. As compared with the wild-type enzyme, no significant change in circular dichroism spectra was observed for the mutant proteins. Thermal unfolding of BlGGT, E398A, E398D, E398K and E398Q followed the two-state unfolding process with a transition point (T m) of 47.7-69.4 °C. Tryptophan fluorescence spectra of the mutant enzymes were different from the wild-type protein in terms of fluorescence intensity. Native BlGGT started to unfold beyond ?1.92 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]0.5, N-U, at 3.07 M equivalent to free energy change ([Formula: see text]) of 14.53 kcal/mol for the N ? U process, whereas the denaturation midpoints for the mutant enzymes were 1.31-2.99 M equivalent to [Formula: see text] of 3.29-12.05 kcal/mol. Taken together, these results strongly suggest that the explored glutamate residue is indeed important for the autocatalytic processing of BlGGT.

SUBMITTER: Chi MC

PROVIDER: S-EPMC3678132 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase.

Chi Meng-Chun MC Chen Yi-Yu YY Lo Huei-Fen HF Lin Long-Liu LL

FEBS open bio 20120928

The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was explored by site-directed mutagenesis. This glutamate was substituted by either alanine, aspartate, arginine or glutamine and the expressed mutant enzymes were purified to apparent homogeneity with metal-affinity chromatography. SDS-PAGE analysis showed that E398A, E398D and E398K were unable to process themselves into a large and a small subunit. However, E398Q was not only a ...[more]

PMID: 23772362

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Project description:The confounding consequences of Helicobacter bilis infection in experimental mice populations are well recognized, but the role of this bacterium in human diseases is less known. Limited data are available on virulence determinants of this species. In Helicobacter pylori, ?-glutamyltranspeptidase (?GT) contributes to the colonization of the gastric mucosa and to the pathogenesis of peptic ulcer. The role of ?GT in H. bilis infections remains unknown. The annotated genome sequence of H. bilis revealed two putative ggt genes and our aim was to characterize these H. bilis ?GT paralogues. We performed a phylogenetic analysis to understand the evolution of Helicobacter ?GTs and to predict functional activities of these two genes. In addition, both copies of H. bilis ?GTs were expressed as recombinant proteins and their biochemical characteristics were analysed. Functional complementation of Esherichia coli deficient in ?GT activity and deletion of ?GT in H. bilis were performed. Finally, the inhibitory effect of T-cell and gastric cell proliferation by H. bilis ?GT was assessed. Our results indicated that one gene is responsible for ?GT activity, while the other showed no ?GT activity due to lack of autoprocessing. Although both H. bilis and H. pylori ?GTs exhibited a similar affinity to L-Glutamine and ?-Glutamyl-p-nitroanilide, the H. bilis ?GT was significantly less active. Nevertheless, H. bilis ?GT inhibited T-cell proliferation at a similar level to that observed for H. pylori. Finally, we showed a similar suppressive influence of both H. bilis and H. pylori ?GTs on AGS cell proliferation mediated by an apoptosis-independent mechanism. Our data suggest a conserved function of ?GT in the Helicobacter genus. Since ?GT is present only in a few enterohepatic Helicobacter species, its expression appears not to be essential for colonization of the lower gastrointestinal tract, but it could provide metabolic advantages in colonization capability of different niches.

Dataset Information

Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis ?-glutamyltranspeptidase.

Publications

Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase.

Similar Datasets

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