Posttranslational Regulation of Nitrogenase Activity by Reversible ADP-Ribosylation; How are the Regulatory Enzymes Drat and Drag Regulated?

Abstract

Nitrogenase activity in Rhodospirillum rubrum, Azospirillum brasilense and related bacteria is regulated by the reversible ADP-ribosylation of arg-101 of the dinitrogenase reductase protein (Ludden and Roberts 1989). Only one of the two arg-101 residues of the NifH homodimer is modified at one time, presumably because of steric hindrance. The ADP-ribosylation of dinitrogenase reductase prevents its productive association with dinitrogenase and thus neither electron transfer nor ATP hydrolysis occurs (Murrell et al. 1988). The NAD-dependent ADP-ribosylation of dinitrogenase reductase is catalyzed by DRAT (Dinitrogenase Reductase ADP-ribosyl Transferase) and the removal of ADP-ribose is catalyzed by DRAG (Dinitrogenase Reductase Activating Glycohydrolase). DRAT and DRAG are encoded by the draTG operon, which is not co-regulated with the nif genes but which is found near the nifHDK operon in R. rubrum, A. lipoferum and A. brasilense (Fitzmaurice et al. 1989; Fu et al. 1989; Fu et al. 1990; Zhang et al. 1992). Both DRAT and DRAG activities are regulated in vivo. Regulation of DRAG was established when 32Plabelled ADP-ribose could not be chased from dinitrogenase reductase during conditions favoring modification (Kanemoto and Ludden 1984). If DRAG was unregulated, the labelled ADP-ribose group would have been continuously replaced (chased) by unlabelled ADP-ribose. tDRAT is regulated in vivo becuase a mutant lacking DRAG but containing DRAT activity accumulated dinitrogenase in the active form until a signal (darkness or ammonium) was given to initiate the modification (Liang et al. 1991). The regulation of both DRAT and DRAG is through inhibition of their activities by other factors, because both enzymes are active in the purified form and require no other factors for activation.