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 i solated from the bacterium Ochrobactrum anthropi. A PCR fragment of 23 0 bp was obtained using oligonucleotide primers deduced from N-termina l and 'internal' sequences of the purified enzyme. The gene was obtain ed by screening of a genomic DNA partial library from O. anthropi cons tructed in pBluescript with a PCR fragment probe. The gene encodes a p rotein (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 overe xpressed product of the gene was also confirmed to have in vivo GST ac tivity towards CDNB. The interaction of the recombinant GST with sever al 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 cata lytic serine residue present in Theta-class GSTs. Substitution of the Ser-ll residue resulted in a mutant OaGST protein lacking CDNB-conjuga ting activity; moreover the mutant enzyme was not able to bind Sepharo se-GSH affinity matrices.