Regulation, structure and function of glutathione reductase and its transcriptional regulator NtcA from the cyanobacterium Anabaena PCC 7120

Abstract: Glutathione reductase (GR) is an important antioxidant enzyme in all aerobic life. Theenzyme was purified from cyanobacterium Anabaena PCC 7120 and overexpressed inEscherichia coli. It has physicochemical and kinetic properties similar to GR fromother sources, but unique features in the pyridine nucleotide-binding site. The secondArg residue required for NADPH binding is absent. In addition, Anabaena GR carriesthe GXGXXG "fingerprint" motif which otherwise characterizes NAD(H)-dependentenzymes. Structural analysis by homology modeling suggested that a Ser residue inthe "fingerprint" motif is important for interaction with NADPH, and an extra loop inthe Anabaena GR near the entrance of pyridine nucleotide-binding site may beresponsible for the lack of affinity for 2', 5'-ADP Sepharose 4B.Mutagenesis studies showed that the loop deletion mutant was a more efficient enzyme with both coenzymes used than the wild-type enzyme, and have higher affinity for the 2, 5'-ADP affinity matrix. In contrast, introduction of the second Arg did not improve either the enzyme activity or the affinity for the Sepharose.The gene encoding Anabaena GR was isolated. It contains two active promoters. One of these is negatively regulated by the transcription factor NtcA under nitrogen fixing condition. In addition, the binding of NtcA to the GR gene promoter is regulated in a redox dependent manner in vitro. This may be significant in heterocysts, in which high-level expressed NtcA may down-regulate GR, since the microaerobic environment established for nitrogen fixation requires less activity of the antioxidant enzyme.NtcA was heterologously expressed in E. coli and was used for the in vitroselection of the NtcA DNA-binding motif. GTA-(N8)-TAC appears to be a morefavorable binding motif than TGT-(N9 or 10)-ACA. A conserved half-site is essential for NtcA binding and only one variable base in the six bases palindromic sequences was allowed. The different recognition consensus sequences probably reflect thediversity in gene regulation under different physiological conditions.