Role of Aggregatibacter actinomycetemcomitans in glutathione catabolism.
BACKGROUND
Our previous studies demonstrated that three enzymes, gamma-glutamyltransferase (GGT), cysteinylglycinase (CGase) and cystalysin, are required for the catabolism of glutathione to produce hydrogen sulfide (H(2)S) in Treponema denticola. In this study, we examined glutathione catabolism in Aggregatibacter actinomycetemcomitans.


METHODS
The GGT and CGase of A. actinomycetemcomitans were determined by biological methods and GGT was characterized using a molecular biological approach.


RESULTS
A. actinomycetemcomitans showed GGT and CGase activity, but could not produce H(2)S from glutathione. The addition of recombinant T. denticola cystalysin, an l-cysteine desulfhydrase, to whole cells of A. actinomycetemcomitans resulted in the production of H(2)S from glutathione. Subsequently, we cloned A. actinomycetemcomitans GGT gene (ggt) and overexpressed the 63 kDa GGT protein. The recombinant A. actinomycetemcomitans GGT was purified and identified. The K(cat)/K(m) of the recombinant GGT from N-gamma-l-glutamyl-4-nitroaniline as substrate was 31/microm/min. The activity of GGT was optimum at pH 6.9-7.1 and enhanced by thiol-containing compounds.


CONCLUSIONS
The results demonstrated that A. actinomycetemcomitans had GGT and CGase activities and that the GGT was characterized. The possible role of A. actinomycetemcomitans in glutathione metabolism and H(2)S production from oral bacteria was discussed.
Department of Orthodontics, University of Texas Health Sciences Center, San Antonio, TX 78229, USA. chul@uthscsa.edu
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D016976:Aggregatibacter actinomycetemcomitans / Q000201:enzymology / Q000378:metabolism*; D000641:Ammonia / Q000032:analysis; D001439:Bacteroides / Q000378:metabolism; D003001:Cloning, Molecular; D003542:Cystathionine gamma-Lyase / Q000378:metabolism; D004150:Dipeptidases / Q000378:metabolism; D004151:Dipeptides / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005051:Eubacterium / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018698:Glutamic Acid / Q000032:analysis; D005973:Glutamine / Q000031:analogs & derivatives / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D006862:Hydrogen Sulfide / Q000032:analysis / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis / Q000378:metabolism; D019289:Pyruvic Acid / Q000032:analysis; D017422:Sequence Analysis, DNA; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D048373:Treponema denticola / Q000201:enzymology; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004151:Dipeptides; D013438:Sulfhydryl Compounds; D005973:Glutamine; C028505:cysteinylglycine; D018698:Glutamic Acid; C001300:gamma-glutamine-4-nitroanilide; D000641:Ammonia; D019289:Pyruvic Acid; D005723:gamma-Glutamyltransferase; C019974:cysteinyl-glycine dipeptidase; D004150:Dipeptidases; D003542:Cystathionine gamma-Lyase; D005978:Glutathione; D006862:Hydrogen Sulfide