Reversible ADP-ribosylation of dinitrogenase reductase forms the basis of posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. This report describes the physiological effects of mutations in the genes encoding the enzymes that add and remove the ADP-ribosyl moiety. Mutants lacking a functional draT gene had no dinitrogenase reductase ADP-ribosyltransferase (DRAT, the draT gene product) activity in vitro and were incapable of modifying dinitrogenase reductase with ADP-ribose in vivo. Mutants lacking a functional draG gene had no dinitrogenase reductase-activating glycohydrolase (DRAG, the draG gene product) activity in vitro and were unable to remove ADP-ribose from the modified dinitrogenase reductase in vivo. Strains containing polar mutations in draT had no detectable DRAG activity in vitro, suggesting likely cotranscription of draT and draG. In strains containing draT and lacking a functional draG, dinitrogenase reductase accumulated in the active form under derepressing conditions but was rapidly ADP-ribosylated in response to conditions that cause inactivation. Detection of DRAT in these cells in vitro demonstrated that DRAT is itself subject to posttranslational regulation in vivo. Mutants affected in an open reading frame immediately downstream of draTG showed regulation of dinitrogenase reductase by ADP-ribosylation, although differences in the rates of ADP-ribosylation were apparent.Rhodospirillum rubrum, a purple, nonsulfur, photosynthetic bacterium, has a well-characterized system of posttranslational regulation of its nitrogenase by ADP-ribosylation. The nitrogenase complex consists of two proteins: dinitrogenase, an a232 tetramer of the nifK and nifD gene products, contains the active site of nitrogenase, and dinitrogenase reductase, an a2 dimer of the nifH gene product, supplies the reducing power to dinitrogenase. Regulation of the complex is achieved by reversible mono-ADP-ribosylation of dinitrogenase reductase at Arg-101 (23) in response to light level and a fixed nitrogen concentration (12).Two enzymes have been shown to perform this regulation in vitro. Dinitrogenase reductase ADP-ribosyltransferase (DRAT, the gene product of dra7) transfers an ADP-ribose from NAD to one subunit of dinitrogenase reductase (18,20), thereby inactivating nitrogenase by preventing electron flow within the nitrogenase complex (22). Dinitrogenase reductase-activating glycohydrolase (DRAG, the gene product of draG) removes the ADP-ribosyl group from dinitrogenase reductase, restoring activity to the nitrogenase complex (21, 28-30). The draT and draG genes have been isolated and sequenced (7) and are adjacent to, but transcribed in the opposite direction from, the nifHDK genes (15).The first description of mono-ADP-ribosylation was that of eucaryotic elongation factor 2 by diphtheria toxin (11). Subsequently, a number of other bacterial toxins, including cholera toxin and pertussis toxin, have been shown to act by ADP-ribosylating proteins in eucaryotic cells (reviewed in * Corresponding aut...

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Dinitrogenase reductase-activating glycohydrolase (DRAG) is responsible for removing the ADP-ribose moiety from post-translationally inactivated nitrogenase of Rhodospirillum rubrum. Using DRAG purified from an overexpressing strain (UR276), further properties of this enzyme were studied, including its u.v.-visible and fluorescence spectra and its stability in air. DRAG appears to require no covalently bound inorganic cofactors for its activity or regulation. Previously, purified DRAG was found to be rapidly inactivated in air. The air-catalysed lability originated with the presence of sodium dithionite and Mn2+ throughout the purification of the enzyme. This lability can be mimicked using H2O2, which is known to oxidatively inactivate proteins containing bivalent metals. Implications for the regulation of nitrogenase are discussed with respect to the lack of sensitivity to air of the regulatory enzyme, DRAG.

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