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J. Biol. Chem., Vol. 283, Issue 28, 19351-19358, July 11, 2008

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

Lianrui Chu¹, Yanlai Lai, Xiaoping Xu, Scott Eddy, Shuang Yang, Li Song, and David Kolodrubetz^¶

From the Departments of Orthodontics, Periodontics, and ^¶Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229

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, -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, -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.

Received for publication, February 7, 2008 , and in revised form, April 28, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant RO1 DE-13819. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: University of Texas Health Science Center at San Antonio, San Antonio, TX 78229. Tel.: 210-567-6269; Fax: 210-567-6858; E-mail: chul{at}uthscsa.edu.

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