Introduction: Our previous studies demonstrated that three enzymes, gamma-glutamyltransferase (GGT), cysteinylglycinase (CGase) and cystalysin, are required for the catabolism of glutathione to produce hydrogen sulfide (H(2)S) in Treponema denticola. In this study, we examined glutathione catabolism in Aggregatibacter actinomycetemcomitans.
Methods: The GGT and CGase of A. actinomycetemcomitans were determined by biological methods and GGT was characterized using a molecular biological approach.
Results: A. actinomycetemcomitans showed GGT and CGase activity, but could not produce H(2)S from glutathione. The addition of recombinant T. denticola cystalysin, an l-cysteine desulfhydrase, to whole cells of A. actinomycetemcomitans resulted in the production of H(2)S from glutathione. Subsequently, we cloned A. actinomycetemcomitans GGT gene (ggt) and overexpressed the 63 kDa GGT protein. The recombinant A. actinomycetemcomitans GGT was purified and identified. The K(cat)/K(m) of the recombinant GGT from N-gamma-l-glutamyl-4-nitroaniline as substrate was 31/microm/min. The activity of GGT was optimum at pH 6.9-7.1 and enhanced by thiol-containing compounds.
Conclusion: The results demonstrated that A. actinomycetemcomitans had GGT and CGase activities and that the GGT was characterized. The possible role of A. actinomycetemcomitans in glutathione metabolism and H(2)S production from oral bacteria was discussed.