Molecular Cloning of the γ‐Glutamyltranspeptidase Gene from a Pseudomonas Strain

Abstract

gamma-Glutamyltranspeptidase (GGT) was purified from a Pseudomonas sp. strain A14. The purified enzyme was found to be composed of two nonidentical subunits with molecular weights of 39,000 and 22,000 and had a pI of > 8.6. The partial N-terminal amino acid sequences of both subunits and some proteolytic fragments were determined. Using mixed oligonucleotides designed from the partial amino acid sequences as hybridization probes, one cosmid clone which contained the GGT gene was isolated from a Pseudomonas sp. strain A14 cosmid genome library, and the DNA sequence of the GGT gene was determined. The nucleotide sequence and the protein sequence analysis revealed that GGT was synthesized as a precursor protein of 575 amino acids and then processed to mature enzyme, presumably after removal of a signal peptide. Comparison of the predicted amino acid sequence of Pseudomonas GGT with published results for Escherichia coli K-12 and rat kidney GGTs shows that the protein sequence of Pseudomonas GGT is 51% and 33% identical to the E. coli and rat GGT sequences, respectively. Higher similarity is observed among the small subunits, which have been thought to have a binding site for the gamma-glutamyl residue. Expression of the cloned Pseudomonas GGT gene in E. coli was subjected to Western blot analysis using antibody raised against the purified GGT. This suggested that processing of the precursor protein to its subunits is temperature-dependent, because the amount of mature GGT protein was increased when the culture was performed at low temperature.

Purification and Properties of Two Isozymes of γ-Glutamyltranspeptidase fromBacillus subtilisTAM-4

“…PGA was prepared from the culture in M medium of Bacillus subtilis TAM-4 by the method previously described.! 7) Bacterial strain and clilture conditions. B. subtilis TAM-4, isolated previously from soiL I 7) was used.…”

“…3) However, it has been reported that GGT is involved in the reclamation of circulating glutathione in conjunction with cysteinyl-glycine dipeptidase. 4 ) On the other hand, GGTs have also been purified from Gram-negativeS 7) and positive 8 . 9 ) bacteria.…”

γ-Glutamyltransferase Is a Helicobacter pylori Virulence Factor but Is Not Essential for Colonization

“…However, GGT expression and activity in bacteria has been poorly characterized. Recently, the ggt gene for H. pylori was identified and sequenced by Chevalier et al (7), making it one of only several bacterial species in which the ggt gene has been characterized (18,34,38). Chevalier et al reported that deletion of GGT had no deleterious effect on the ability of H. pylori to grow in culture, but GGT is essential for H. pylori infection of the mouse (7).…”

Identification, sequence, and expression of the gene encoding gamma-glutamyltranspeptidase in Bacillus subtilis

“…The physiological significance of the enzyme is largely unknown, but it is the only protease known to cleave intact glutathione, and it has been used as a clinical marker of liver damage and hepatocarcinogenesis (5,7,9). GGT-encoding genes from mammalian and bacterial species have been sequenced (3,8,12,14,17,27). The proteins have extensive homology and are translated as propolypeptides of approximately 580 amino acids which are subsequently processed to form the two subunits of the mature enzymes.…”