Abstract
Addition of ammonium to the cultures of Synechococcus sp. PCC7942 reversibly inhibits the activity of nitrate/nitrite transporter (NRT). NRT of the cyanobacterium is an ABC transporter, one of the ATP-binding subunits of which (NrtC) has a large C-terminal domain (R domain) required for the ammonium-promoted inhibition of NRT. To gain insight into the molecular mechanism of the regulation of NRT, we expressed the R domain as a His-tagged protein in E. coli and characterized it biochemically. Equilibrum dialysis experiments showed that the recombinant R domain specifically binds 2-oxoglutarate (2-OG) with a dissociation constant of ~190μM. The intracellular 2-OG concentration of Synechococcus sp. PCC7942 was determinded to be ~150μM in nitrate- or nitrite-grown cells, which was decreased rapidly to ~10μM after addition of ammonium. These results suggested that 2-OG is likely to affect NRT activity by binding to and dissociating from the R domain in vivo.
