Glutathione reductase from Escherichia coli: cloning and sequence analysis of the gene and relationship to other flavoprotein disulfide oxidoreductases.

A glutathione reductase negative strain of Escherichia coli K-12 was isolated as a thermoresistant survivor when a gor::MuctsAp lysogen was subjected to elevated temperature. It was found that in addition to being ampicillin sensitive this mutant was hypersensitive to arsenate, which may be connected with the fact that the gor gene maps between 77 and 78 min on the E. coli genome, close to the pit locus encoding the major arsenate transport system of E. coli. A derivative of this mutant was used as the recipient in a screen of the Clarke and Carbon hybrid plasmid bank of E. coli DNA. A plasmid, pGR, was isolated that encodes both an arsenate-resistance element and glutathione reductase. Restriction mapping of this plasmid showed that the insert DNA is approximately 10 kilobase pairs in length, and a fragment of the gor gene was identified that allowed the gor gene to be accurately mapped on pGR by a combination of restriction analysis and Southern blotting. The DNA sequence of the gor gene was determined and found to encode a protein of 450 amino acid residues. The glutathione reductase of E. coli is very homologous to the human enzyme and is also related (though less closely) to other flavoprotein disulfide oxidoreductases whose sequences are available. These enzymes have retained a common mechanism while evolving different specificities.