Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi

Abstract

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.

BphK shows dechlorination activity against 4-chlorobenzoate, an end product ofbph-promoted degradation of PCBs

“…The Bioscreen C 0 automated turbidimetric instrument can run 200 samples at a time making this method e⁄cient for screening large numbers of samples. This assay, for example, would be extremely advantageous in mutagenesis studies that are routinely used to study GST structure and function [20] or screening for novel substrates as described in this report.…”

Expression of glutathioneS-transferase and peptide methionine sulphoxide reductase inOchrobactrum anthropiis correlated to the production of reactive oxygen species caused by aromatic substrates

“…All experiments were carried out with O. anthropi [17,18]. The strain was cultivated in Luria-Bertani broth.…”

The Impact of Nitric Oxide Toxicity on the Evolution of the Glutathione Transferase Superfamily

“…In particular, Tyr-GST: hGSTA1-1 (12), hGSTM2-2 (12), hGSTP1-1 (12), hGSTA2-2 (15), and hGSTA3-3 (16), O. volvulus GST2 (17), Schistosoma hematobium GST (18), P. falciparum GST (19), and squid GST (20); Ser-GSTs: hGSTT2-2 (21), Phi 1 GST Mais (22), hGSTZ (23), Anopheles dirus GSTD3-3 (24), A. dirus GSTD4-4 (25), A. dirus GSTD5-5 (26); Cys-GSTs: hGSTO1-1 (27) Proteus mirabilis GSTB1-1 (12), Sphingomonas paucimobilis GST (supplemental Methods), Burkholderia xenovorans GST (28), Ochrobactrum anthropi GST (29), and Coccidioides immitis GST (30). Mutant GSTs were expressed and purified as follows: Y7F hGSTP1-1 (31), Y7C hGSTP1-1 (31), S11A hGSTT2-2 (32), C10A GST from P. mirabilis (33), and C10A GST from O. anthropi (34).…”