Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi.
The gene coding for a novel glutathione S-transferase (GST) has been isolated from the bacterium Ochrobactrum anthropi. A PCR fragment of 230 bp was obtained using oligonucleotide primers deduced from N-terminal and 'internal' sequences of the purified enzyme. The gene was obtained by screening of a genomic DNA partial library from O. anthropi constructed in pBluescript with a PCR fragment probe. The gene encodes a protein (OaGST) of 201 amino acids with a calculated molecular mass of 21738 Da. The product of the gene was expressed and characterized; it showed GST activity with substrates 1-chloro-2, 4-dinitrobenzene (CDNB), p-nitrobenzyl chloride and 4-nitroquinoline 1-oxide, and glutathione-dependent peroxidase activity towards cumene hydroperoxide. The overexpressed product of the gene was also confirmed to have in vivo GST activity towards CDNB. The interaction of the recombinant GST with several antibiotics indicated that the enzyme is involved in the binding of rifamycin and tetracycline. The OaGST amino acid sequence showed the greatest identity (45%) with a GST from Pseudomonas sp. strain LB400. A serine residue in the N-terminal region is conserved in almost all known bacterial GSTs, and it appears to be the counterpart of the catalytic serine residue present in Theta-class GSTs. Substitution of the Ser-11 residue resulted in a mutant OaGST protein lacking CDNB-conjugating activity; moreover the mutant enzyme was not able to bind Sepharose-GSH affinity matrices.
Favaloro
B
B
Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 'G. Paone' Environmental Health Center, Department of Environmental Sciences, 66030 Santa Maria Imbaro, Italy. favaloro@cmns.mnegri.it
Tamburro
A
A
Angelucci
S
S
Luca
A D
AD
Melino
S
S
di Ilio
C
C
Rotilio
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015698:Genomic Library; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D007527:Isoenzymes; D005982:Glutathione Transferase