A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism

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. H 2 S production in this organism has been proposed to occur via a three enzyme pathway, ␥-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. . Importantly, in combination with the two other previously purified T. denticola enzymes, ␥-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H 2 S. These results prove that T. denticola contains the entire three-step pathway to produce H 2 S from glutathione, which may be important for pathogenesis.The volatile sulfur compound H 2 S can be produced by the metabolic activity of numerous oral bacteria, including several periodontal pathogens (1-3). This gas, which is malodorous and highly toxic (4 -6), is found in high concentrations in periodontal pockets (7-9) and may play a role in some of the tissue destruction seen in periodontal diseases (7,8,10). H 2 S can be produced from the metabolism of several molecules, but glutathione (L-␥-glutamyl-L-cysteinylglycine) is believed to be the major source for H 2 S production in the oral cavity; human cells, especially polymorphonuclear leucocytes, have high concentrations (up to 4 mM) of glutathione that can be released when host cells are damaged in the periodontal pocket. Although a number of oral bacteria have been tested, only a few of them are able to catabolize glutathione into H 2 S (3, 11, 12). Treponema denticola, which appears to play a significant role in the development of acute and chronic periodontal diseases in humans (13-17), is the only oral pathogen in which the proteins involved in this catabolic pathway have begun to be identified and characterized (18 -22). Glutathione catabolism to H 2 S (and glutamate, glycine, ammonia, and pyruvate) has been proposed to occur via a three-step enzyme pathway in this spirochete (12). In the first step, glutathione is split into glutamate and Cys-Gly. This dipeptide is then hydrolyzed into glycine and L-cysteine followed by the breakdown of L-cysteine into pyruvate, ammonia, and H 2 S. The enzymes involved in the first and third steps have been purified from T. denticola and characterized (19 -30). ␥-Glutamyltransferase (GGT) 2 is a 27-kDa protein that catalyzes the cleavage of glutathione into glutamate ...

Virulence Factors of the Oral Spirochete Treponema denticola

“…Relatively little is known regarding the metabolism of T. denticola, and many of its metabolic pathways are likely to be novel (Rother et al, 2001;Seshadri et al, 2004;Chu et al, 2008). It primarily utilizes serine, alanine, cysteine, and glycine when grown in vitro and generates fermentation products including acetate, lactate, succinate, formate, pyruvate, ethanol, carbon dioxide, hydrogen sulfide (H 2 S), and ammonia (Hespell and Canale-Parola, 1971).…”

“…H 2 S is cytotoxic for a variety of host cells, including gingival fibroblasts and epithelial cells (Beauchamp et al, 1984;Reiffenstein et al, 1992;Chu et al, 1999;Yoshimura et al, 2000). T. denticola is the only known oral bacterium to contain the three-step pathway required to produce H 2 S, from the abundant host tripeptide, glutathione (Chu et al, 2008). This pathway contains cysteinylglycinase, γ-glutamyltransferase, and the novel enzyme, cystalysin (Makinen and Makinen, 1997;Chu et al, 2002).…”

Gene Regulation, Two Component Regulatory Systems, and Adaptive Responses in Treponema Denticola

“…Important to both metabolism and virulence, T. denticola strains encode an array of 18-25 proteases. Two proteases that have been experimentally characterized in some detail are dentilisin (Chi et al 2003;Ellen et al 2000;Ishihara et al 1998;McDowell et al 2009;Miller et al 2016;Yamazaki et al 2006) and cystalysin (Chu et al 2002(Chu et al , 2008Krupka et al 2000;Zhang et al 2010). Both play important roles in T. denticola pathogenesis.…”

Characterization of anAspergillus oryzaeCysteinyl Dipeptidase Expressed inEscherichia coli

“…Some aminopeptidases have been reported to have activity toward Cys-Gly, 11) and some leucine aminopeptidases in the M17 family have high activity toward Cys-Gly. 5,12) The optimal pH range of CdpA for hydrolyzing CysGly was pH 7-8 (Fig. 1A), and CdpA was stable in a pH range of 6-9 (Fig.…”