Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis.
The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421-425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli sigma 70 consensus promoter and to promoters of P. mirabilis cat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-beta-D-thiogalactopyranoside and growth at 37 degrees C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.
Perito
B
B
Dipartimento di Biologia Animale e Genetica, Università di Firenze, Italy.
Allocati
N
N
Casalone
E
E
Masulli
M
M
Dragani
B
B
Polsinelli
M
M
Aceto
A
A
Di Ilio
C
C
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D017931:DNA Primers; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011513:Proteus mirabilis / Q000187:drug effects / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004137:Dinitrochlorobenzene; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D005982:Glutathione Transferase