Characterization of three γ-glutamyltranspeptidases from Pseudomonas aeruginosa PAO1

The Pseudomonas aeruginosa strain, PAO1, has three putative gamma-glutamyltranspeptidase (GGT) genes: ggtI, ggtII, and ggtIII. In this study, the expression of each of these genes in P. aeruginosa PAO1 was analyzed, and the properties of the cor-responding GGT proteins were investigated. This is the first report on biochemical characterization of GGT paralogs from Pseudomonas species. The crude extracts prepared from P. aeruginosa PAO1 exhibited hydrolysis and transpeptidation activities of 17.3 and 65.0 mU/mg, respectively, and the tran-scription of each gene to mRNA was confirmed by RT-PCR. All genes were cloned, and the expression plasmids constructed were introduced into an Escherichia coli expression system. Enzyme activity of the expressed protein of ggtI (PaGGTI) was not detected in the system, while the enzyme activities of the expressed proteins derived from ggtII and ggtIII (PaGGTII and PaGGTIII, respectively) were detected. However, the enzyme activity of PaGGTII was very low and easily decreased. PaGGTII with C-terminal his-tag (PaGGTII25aa) showed increased activity and stability, and the purified enzyme con -sisted of a large subunit of 40 kDa and a small sub-unit of 28 kDa. PaGGTIII consisted of a large sub-unit of 37 kDa and a small subunit of 24 kDa. The maximum hydrolysis and transpeptidation activities of PaGGTII25aa were obtained at 40 degrees C-50 degrees C, and the maximum hydrolysis and transpeptidation activities of PaGGTIII were obtained at 50 degrees C-60 degrees C. These enzymes retained approximately 80% of their hydrolysis and transpeptidation activities after incu-bation at 50 degrees C for 10 min, reflecting good stability. Both PaGGTII25aa and PaGGTIII showed higher activities of hydrolysis and transpeptidation in the alkali range than in the acidic range. However, they were highly stable at a wide pH range (5-10.5).

Automated summary

This study analyzed the expression of three putative gamma-glutamyltranspeptidase (GGT) genes in Pseudomonas aeruginosa PAO1 and investigated the properties of the corresponding GGT proteins. It is the first report on the biochemical characterization of GGT paralogs from Pseudomonas species.