Role for recombinant gamma-glutamyltransferase from Treponema denticola in glutathione metabolism.
Volatile sulfur compounds, including hydrogen sulfide (H(2)S), have been implicated in the development of periodontal disease. Glutathione is an important thiol source for H(2)S production in periodontal pockets. Our recent studies have delineated a pathway of glutathione metabolism in Treponema denticola that releases H(2)S. In this pathway, gamma-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-gamma-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 H(2)S, ammonia, and pyruvate. This was accompanied by the suppression of hemoxidative and hemolytic activities of the bacteria. Purified GGT was inactivated by TLCK (Nalpha-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 H(2)S, 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.
Chu
Lianrui
L
Department of Orthodontics, University of Texas Health Science Center, San Antonio, Texas 78229, USA. chul@uthscsa.edu
Xu
Xiaoping
X
Dong
Zheng
Z
Cappelli
David
D
Ebersole
Jefferey L
JL
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D005978:Glutathione / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism; D016966:Porphyromonas gingivalis / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014208:Treponema / Q000201:enzymology* / Q000235:genetics; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D006418:Heme; D010088:Oxidoreductases; D005723:gamma-Glutamyltransferase; D005978:Glutathione