gamma-Glutamyltransferase from the outer cell envelope of Treponema denticola ATCC 35405

Abstract

The human oral spirochete Treponema denticola ATCC 35405 was shown to exhibit relatively high enzyme activity toward the ␥-glutamyl amide bond present in N-␥-L-glutamyl-4-nitroaniline. The enzyme responsible for this catalysis (␥-glutamyltransferase [GGT]; EC 2.3.2.2) was purified by means of fast protein liquid chromatography to two sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-pure forms from a mild (0.1%) Triton X-100 extract of washed cells. The GGT was studied primarily with regard to its hydrolytic activity by using N-␥-L-glutamyl-4-nitroaniline as a substrate, although the GGT was shown to catalyze transpeptidation reactions. The high-molecular-mass form of the GGT gave a value of about 213 kDa by SDS-PAGE when heat treatment was omitted and one of 26 kDa after heat treatment; mass spectrometry gave a value of 26.877. The larger form may represent an aggregate with nonprotein structures (possibly of a carbohydrate nature). The preliminary N-terminal sequence of the GGT is MKKPLIGITGSXLYETSQXXF. The enzyme was highly active on glutathione, transferring its Glu residue either to a water molecule or to the Gly-L-Leu dipeptide. The GGT stability was absolutely dependent on the presence of free thiol(s), while no evidence of metalloenzyme nature was obtained. The proposed location of the GGT in the outer cell envelope and its high activity on glutathione, a major nonprotein thiol present in virtually all cells, suggest that the GGT may play a role in the propagation of T. denticola within inflamed periodontal tissues.

Crevicular fluid glutathione levels in periodontitis and the effect of non‐surgical therapy

“…In reality, glutathione levels detected in GCF, like those detected in ALF, are likely to depend upon a variety of factors including the rate of synthesis and export by epithelium ( Fig. 3 demonstrates synthesis and fates of intracellular GSH), redox state of intracellular glutathione, size of the glutathione pool able to undergo redox cycling, the presence of GSH-reactive species in the crevice (Ballatori et al 2009, Biswas & Rahman 2009 and the metabolism of GSH by certain bacteria (certain Fusobacteria, Peptostreptococcus micros and Treponema denticola) to hydrogen sulphide (Persson et al 1990, Carlsson et al 1993, Makinen & Makinen 1997, Chu et al 2003, Zappacosta et al 2007). Indeed, distinct metabolic pathways underlying this latter process in T. denti-cola have been reported (Chu et al 2002).…”

The role of reactive oxygen and antioxidant species in periodontal tissue destruction

“…The discovery of millimolar levels of GSH in gingival crevicular fluid (83) and high levels contributing to the total antioxidant status of the cervical epithelium (93), has led to the hypothesis that GSH may represent an innate and fundamental defense strategy at exposed epithelial surfaces (79, 83). Interestingly, some periodontal pathogens (certain Fusobacteria , Peptostreptococcus micros , and Treponema denticola ) metabolize GSH and convert it to the cytotoxic hydrogen sulfide (69, 87, 256, 318), and recently, distinct metabolic pathways underlying this process in T. denticola were reported (86).…”

“…used GSH to generate hydrogen sulfide. It has been proposed that the enzyme γ ‐glutamyl‐transpeptidase, which breaks GSH down to cys‐gly in human cell membranes, is also present in T. denticola outer cell envelopes and that γ ‐glutamyl‐transpeptidase may play a role in the propagation of T. denticola within inflamed periodontal tissues (256). Chu et al.…”

The antioxidant master glutathione and periodontal health

“…Mäkinen and Mäkinen[ 131 ] reported the presence of enzyme GGT (that breaks down glutathione to Cys-Gly in human cell membrane) in the outer cell envelope of T. denticola that may play an important role in its propagation in inflamed periodontal tissues. Three-step pathways have been proposed for the glutathione metabolism in T. denticola .…”