CLONING, SEQUENCING, AND REGULATION OF THE GLUTATHIONE-REDUCTASE GENEFROM THE CYANOBACTERIUM ANABAENA PCC-7120

Glutathione reductase (GR) was purified from the cyanobacterium Anabae na PCC 7120. A 3-kilobase genomic DNA fragment containing the coding s equence for the GR gene (gor) was identified and cloned by polymerase chain reaction based on sequences of selected peptides isolated from p roteolyzed GR. The coding sequence encompassing 458 amino acid residue s, as well as 360 base pairs of the 5'-flanking region and 430 base pa irs of the 3'-flanking region, were determined. Genomic Southern analy sis indicates that gor is a single-copy gene. A gor antisense RNA prob e hybridized with a 1.4-kilobase transcript, suggesting that the gene is not part of an operon including additional genes. The deduced GR am ino acid sequence shows 41 to 48% identity with those of human. Escher ichia coli, Pseudomonas aeruginosa, pea, and Arabidopsis thaliana GR. The coding sequence of GR was overexpressed in a GR-deficient E., coli strain, SG5, and the recombinant protein was purified. Anabaena GR is NADPH-linked, but a Lys residue replaces an Arg residue involved in N ADPH binding in GR from other species. In addition, Anabaena GR carrie s the GXGXXG ''fingerprint'' motif which otherwise characterizes NAD(H )-dependent enzymes. These differences may contribute to the lack of a ffinity for 2',5'-ADP-Sepharose 4B of Anabaena GR. Three E. coli-type promoter sequences and a BifA/NtcA binding motif were found upstream o f the open reading frame. The middle and the proximal promoters were s hown to be active. However, the use of the middle promoter was depende nt on the nitrogen source in the culture medium. Both GR activity and GR protein concentration increased in ammonium grown cultures in which both the middle and proximal promoters were used for transcriptional initiation. The BifA/NtcA-binding site overlaps the middle promoter se quence and may thus be involved in regulation of differential transcri ption.