A 52-kDa leucyl aminopeptidase from treponema denticola is a cysteinylglycinase that mediates the second step of glutathione metabolism

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, γ- 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/K_m of 8.2 μM^-1 min^-1) not L-Leu-p-NA (k_cat/K_m of 1.1 μM^-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, γ-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.