Metabolic Regulation of Nitrogenase: Regulation and Localization of DRAG (Dinitrogenase Reductase Activating Glycohydrolase)

Abstract

In the photosynthetic bacteria, Rhodospirillum rubrum and Rhodobacter capsulatus nitrogenase is regulated on a metabolic as well as a genetic level. This regulation has also been observed in Azospirillum brasilense and A. lipoferum. By adding switch-off effectors like ammonia, glutamine or subjecting the cells to darkness or anaerobiosis rspectively, dinitrogenase reductase is reversibly inactivated. Addition of NAD+ to R. rubrum give a similar switch-off effect. The molecular event causing this inactivation has been shown by Ludden and coworkers to be a mono-ADPribosylation of dinitrogenase reductase on arg-101. DRAT (dinitrogenase reductase ADP-ribosyl transferase) catalyzes the modification in an ADP and NAD+ dependent reaction. Activation, removal of the ADP-ribose, is catalyzed by DRAG, (dinitrogenase reductase activating glycohydrolase), in a reaction requiring ATP and a cation. Both DRAG and DRAT (draT and draG genes are cotranscribed and the proteins are expressed under both non-nitrogen fixing and nitrogen fixing conditions) undergoes posttranslational regulation (Ludden, Roberts 1989). In crude cell extracts, DRAG is membrane associated but can be washed off the chromatophores with 0.5M NaCl (Nordlund et al 1977). The sequence of DRAG does not reveal any strong hydrophobic regions within the protein which together with the salt effect suggest that it is a soluble protein. The mechanism of the regulation of DRAG is as yet unknown. The redox and nitrogen status of the cell have been suggested as internal signals for regulation of DRAG. Our working hypothesis for the regulation of DRAG involves the association of DRAG to a protein or protein complex within the chromatophore membrane. In previous studies we have shown that MgGDP, but not MgGTP, MgATP or MgADP, release active DRAG from the membranes, indicating that the association in fact is specific (Noren &Nordlund, unpublished).