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

Lianrui Chu, Yanlai Lai, Xiaoping Xu, Scott Eddy, Shuang Yang, Li Song, David Kolodrubetz

Research output: Contribution to journalArticle

28 Citations (Scopus)

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, 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/Km of 8.2 μM-1 min-1) not L-Leu-p-NA (kcat/Km 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.

Original languageEnglish (US)
Pages (from-to)19351-19358
Number of pages8
JournalJournal of Biological Chemistry
Volume283
Issue number28
DOIs
StatePublished - Jul 11 2008

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Treponema denticola
cysteinylglycine
Leucyl Aminopeptidase
Metabolism
Glutathione
Proteins
High pressure liquid chromatography
Electrospray ionization
Hydrogen Sulfide
Metalloproteases
Pathogens
Enzymes
Recombinant Proteins
Escherichia coli
Purification
Mass spectrometry
Hydrolysis
Genes
Amino Acid Sequence Homology
Tissue

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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A 52-kDa leucyl aminopeptidase from treponema denticola is a cysteinylglycinase that mediates the second step of glutathione metabolism. / Chu, Lianrui; Lai, Yanlai; Xu, Xiaoping; Eddy, Scott; Yang, Shuang; Song, Li; Kolodrubetz, David.

In: Journal of Biological Chemistry, Vol. 283, No. 28, 11.07.2008, p. 19351-19358.

Research output: Contribution to journalArticle

Chu, Lianrui ; Lai, Yanlai ; Xu, Xiaoping ; Eddy, Scott ; Yang, Shuang ; Song, Li ; Kolodrubetz, David. / A 52-kDa leucyl aminopeptidase from treponema denticola is a cysteinylglycinase that mediates the second step of glutathione metabolism. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 28. pp. 19351-19358.
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AU - Song, Li

AU - Kolodrubetz, David

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AB - 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/Km of 8.2 μM-1 min-1) not L-Leu-p-NA (kcat/Km 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.

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