DNA Sequence of γ-Glutamyl transpeptidase from Actinobacillus actinomycetemcomitans Y4

A γ-glutamyl peptide-hydrolysing enzyme obtained from A. actinomycetemcomitans Y4 hydrolyses a substrate for γ-glutamyl transpeptidase (GGT) and the enzyme activity is specifically inactivated by γ-glutamyl affinity reagents. But some properties of the bacterial enzyme are different from those of other GGTs. Objectives: In order to compare the predicted amino acid sequence and DNA sequence of the bacterial enzyme with those of other GGTs, we undertook the subcloning and sequencing of the corresponding gene. Methods: Bacterial cells were cultured at 37 °C for 20 h in an atmosphere of 10 % CO₂ and 90% in BHI broth. Bacterial cells were harvested, washed with buffered saline and then treated with lysozyme and RNase. The cells were lysised with SDS and digested with proteinase K. Total DNA was extracted with phenol and chloroform. PCR primers were designed on the basis of DNA sequence of A. actinomycetemcomitans HK1651 GGT gene. Amplification products were electrophoresised in a 2% agarose gel. The PCR product was inserted pETBlue-1 Vector with Perfectly Blunt Cloning Kits. DNA sequencing was analyzed by using ABI PRISM 310NT genetic analyzer. Results: The amino acid sequences of the bacterial enzyme and E. coli K12 GGT were 58.6 % similar. Also higher similarity (64.7 %) was observed between the small subunits which γ-glutamyl-binding subsites localize. Alignment of the DNA sequences of the bacterial enzyme and E. coli K12 GGT revealed identity of 71.8 %. Evolutionary tree constructed by UPGMA method indicated that the bacterial enzyme is more closely related to E. coli K12 GGT than to F. nucleatum GGT and S.aureus. Conclusions: It was suggested that γ-glutamyl peptide-hydrolysing enzyme which we reported, might be GGT-like enzyme. However further investigations, such as expression of the GGT gene, are necessary.