Cloning, sequencing, and regulation of the glutathione reductase gene from the cyanobacterium Anabaena PCC 7120.
Glutathione reductase (GR) was purified from the cyanobacterium Anabaena PCC 7120. A 3-kilobase genomic DNA fragment containing the coding sequence for the GR gene (gor) was identified and cloned by polymerase chain reaction based on sequences of selected peptides isolated from proteolyzed GR. The coding sequence encompassing 458 amino acid residues, as well as 360 base pairs of the 5'-flanking region and 430 base pairs of the 3'-flanking region, were determined. Genomic Southern analysis indicates that gor is a single-copy gene. A gor antisense RNA probe hybridized with a 1.4-kilobase transcript, suggesting that the gene is not part of an operon including additional genes. The deduced GR amino acid sequence shows 41 to 48% identity with those of human, Escherichia 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 NADPH binding in GR from other species. In addition, Anabaena GR carries the GXGXXG "fingerprint" motif which otherwise characterizes NAD(H)-dependent enzymes. These differences may contribute to the lack of affinity for 2',5'-ADP-Sepharose 4B of Anabaena GR. Three E. coli-type promoter sequences and a BifA/NtcA binding motif were found upstream of the open reading frame. The middle and the proximal promoters were shown to be active. However, the use of the middle promoter was dependent 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 sequence and may thus be involved in regulation of differential transcription.
Jiang
F
F
Department of Biochemistry, Uppsala University, Sweden.
Hellman
U
U
Sroga
G E
GE
Bergman
B
B
Mannervik
B
B
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology* / Q000235:genetics*; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005980:Glutathione Reductase / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D003062:Codon; D017931:DNA Primers; D005980:Glutathione Reductase