Abstract
The metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, gamma-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (k cat/Km of 8.2 microm(-1) min(-1)) not l-Leu-p-NA (k cat/Km of 1.1 microm(-1) min(-1)). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, gamma-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis.
Highlights
Thione (L-␥-glutamyl-L-cysteinylglycine) is believed to be the major source for H2S production in the oral cavity; human cells, especially polymorphonuclear leucocytes, have high concentrations of glutathione that can be released when host cells are damaged in the periodontal pocket
The substrate specificity of T. denticola CGase was compared with that of leucine aminopeptidase (LAP) from porcine kidney (Sigma). Both of the substrates were hydrolyzed by both enzymes, the results clearly showed that Cys-Gly was the better substrate for the T. denticola CGase and that Leu-p-NA was the better substrate for porcine LAP (Fig. 4)
The CGase from T. denticola is a 52-kDa protein that is a member of the M17 family of leucyl aminopeptidases
Summary
All of the chemicals and reagents were obtained from Sigma. All of the bacteria used in this study were cultured anaerobically in a Coy anaerobic chamber (5% CO2, 10% H2, and 85%N2) at 37 °C in GM-1 broth [33] supplemented with 3.4% rabbit serum
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