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Infection and Immunity, January 2003, p. 335-342, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.335-342.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Role for Recombinant -Glutamyltransferase from Treponema denticola in Glutathione Metabolism

Lianrui Chu,^1* Xiaoping Xu,¹ Zheng Dong,^2, David Cappelli,¹ and Jefferey L. Ebersole³

Departments of Orthodontics, Periodontics, and Pathology,¹ Community Dentistry, University of Texas Health Science Center, San Antonio, Texas 78229,² Center for Oral Health Research, University of Kentucky, Lexington, Kentucky 40536³

Received 29 July 2002/ Returned for modification 4 September 2002/ Accepted 7 October 2002

Volatile sulfur compounds, including hydrogen sulfide (H₂S), have been implicated in the development of periodontal disease. Glutathione is an important thiol source for H₂S production in periodontal pockets. Our recent studies have delineated a pathway of glutathione metabolism in Treponema denticola that releases H₂S. In this pathway, -glutamyltransferase (GGT) has been proposed to catalyze the first step of glutathione degradation. We have cloned the gene of GGT from T. denticola, which contains an open reading frame of 726 bp encoding a protein of 241 amino acids. Transformation of this gene into Escherichia coli led to the expression of a recombinant protein. After purification by chromatography, the recombinant protein showed enzymatic activity typical of GGT, catalyzing the degradation of Na--glutamyl-4-nitroaniline (GNA) and the hydrolysis of glutathione, releasing glutamic acid or glutamine and cysteinylglycine. L-Cysteine is not a substrate of GGT. Importantly, GNA, when added to T. denticola, was able to compete with glutathione and inhibit the production of H₂S, ammonia, and pyruvate. This was accompanied by the suppression of hemoxidative and hemolytic activities of the bacteria. Purified GGT was inactivated by TLCK (N-p-tosyl-L-lysine chloromethyl ketone) and proteinase K treatment. However, higher enzymatic activity was demonstrated in the presence of 2-mercaptoethanol and dithiothreitol. Our further experiments showed that the addition of recombinant GGT to Porphyromonas gingivalis, a bacterium without significant glutathione-metabolizing capacity, drastically increased the utilization of glutathione by the bacterium, producing H₂S, ammonia, and pyruvate. This was again accompanied by enhanced bacterial hemoxidative and hemolytic activities. Together, the results suggest an important role for GGT in glutathione metabolism in oral bacteria.

Editor: J. D. Clements

Present address: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912.

This article has been cited by other articles:

• Chu, L., Lai, Y., Xu, X., Eddy, S., Yang, S., Song, L., Kolodrubetz, D. (2008). A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism. J. Biol. Chem. 283: 19351-19358 [Abstract] [Full Text]  
• Lai, Y., Chu, L. (2008). Novel Mechanism for Conditional Aerobic Growth of the Anaerobic Bacterium Treponema denticola. Appl. Environ. Microbiol. 74: 73-79 [Abstract] [Full Text]