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, 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.
Chu
Lianrui
L
Department of Orthodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA. chul@uthscsa.edu
Lai
Yanlai
Y
Xu
Xiaoping
X
Eddy
Scott
S
Yang
Shuang
S
Song
Li
L
Kolodrubetz
David
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004150:Dipeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial / Q000502:physiology; D005978:Glutathione / Q000096:biosynthesis*; D006862:Hydrogen Sulfide / Q000378:metabolism; D006868:Hydrolysis; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010518:Periodontitis / Q000201:enzymology / Q000235:genetics / Q000382:microbiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D048373:Treponema denticola / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004151:Dipeptides; D011994:Recombinant Proteins; C028505:cysteinylglycine; D005723:gamma-Glutamyltransferase; D007931:Leucyl Aminopeptidase; C019974:cysteinyl-glycine dipeptidase; D004150:Dipeptidases; D005978:Glutathione; D006862:Hydrogen Sulfide