Nitrogen fixation activity in the photosynthetic bacterium Rhodospirillum rubrum is controlled by the reversible ADP-ribosylation of the dinitrogenase reductase component of the nitrogenase enzyme complex. This report describes the cloning and characterization of the genes encoding the ADP-ribosyltransferase (draT) and the ADP-ribosylglycohydrolase (draG) involved in this regulation. These genes are shown to be contiguous on the R. rubrum chromosome and highly linked to the nifHDK genes. Sequence analysis revealed the use of TTG as the initiation codon of the draT gene as well as a potential open reading frame immediately downstream of draG. The mono-ADP-ribosylation system in R. rubrum is the first in which both the target protein and modifying enzymes as well as their structural genes have been isolated, making it the model system of choice for analysis of this post-translational regulatory mechanism.
Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversib[y regulated by interconversion of the Fe protein between a modified and an unmodified form. Since the discovery of the activation process in 1976, investigators have been unable to demonstrate the inactivation (modification) reaction in vitro. In this study, NAD-dependent modification and concomitant inactivation of the Fe protein were demonstrated in crude extracts of R. rubrum. Activation of the in vitro-modified Fe protein by activating enzyme and structural similarity between the in vivo and in vitro modifications are presented as evidence that the in vitro modification is the physiologically relevant ADP-ribosylation reaction. Using a partially purified preparation, we showed that the inactivating enzyme activity is stimulated by divalent metal ions and ADP, that 02-denatured Fe protein will not serve as a substrate, and that dithionite inhibits the modification reaction.
The mechanism by which MgADP stimulates the activity of dinitrogenase reductase ADP-ribosyltransferase (DRAT) has been examined by using dinitrogenase reductases from Rhodospirillum rubrum, Klebsiella pneumoniae, and Azotobacter vinelandii as acceptor substrates. In the presence of 0.2 mM NAD, maximal rates of ADP-ribosylation of all three acceptors were observed at an ADP concentration of 150 microM; in the absence of added ADP, DRAT activity with the dinitrogenase reductases from R. rubrum and K. pneumoniae was less than 5% of the maximal rate, but the A. vinelandii protein was ADP-ribosylated at 40% of the maximal rate. Of eight dinucleotides tested, only ADP, 2'-deoxy-ADP, and ADP-beta S served as activators of the DRAT reaction; ADP, 2'-deoxy-ADP, and ADP-beta S were also the only dinucleotides found which inhibited acetylene reduction activity by dinitrogenase reductase. The dinucleotide specificities for both DRAT activation and acetylene reduction inhibition were the same for all three dinitrogenase reductases. In the DRAT reaction with the dinitrogenase reductases from K. pneumoniae and A. vinelandii, the Km for NAD was 30-fold higher in the absence of ADP than in its presence; the Km for NAD with the R. rubrum acceptor was not measurable. In the presence of saturating ADP, ADP-ribosylation of dinitrogenase reductase from R. rubrum was inhibited 63% by 1.5 mM ATP. It is concluded that MgADP stimulates DRAT activity by lowering the Km for NAD and that MgADP exerts its effect by binding to dinitrogenase reductase. MgATP inhibits DRAT activity by competing with MgADP for binding to dinitrogenase reductase.
RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from Gα and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a Gα i1 mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. Gα i1 (R178M/A326S) GTPase activity was stimulated 6-to 12-fold by RGS proteins known to act on Gα i subunits and not affected by those unable to act on Gα i , demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of Gα i1 (R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on Gα i1 (R178M/A326S), the authors developed a Transcreener fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gα i1 (R178M/A326S) with a homogeneous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling. (Journal of Biomolecular
The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A [39]) has been well studied in the purple nonsulfur photosynthetic bacterium Rhodospirillum rubrum. In R. rubrum, the loss of cellular nitrogenase activity during switch-off by NH4Cl is correlated with the covalent modification and resulting inactivation of dinitrogenase reductase (13). The covalent modification consists of the ADP-ribosylation of dinitrogenase reductase at . This reaction is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DRAT) (18,19). Dinitrogenase reductase is composed of two identical subunits, and the ADP-ribosylated subunit migrates more slowly than the unmodified subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Dinitrogenase reductase-activating glycohydrolase (DRAG) catalyzes the removal of the ADP-ribose group from the inactive dinitrogenase reductase, thereby reactivating the dinitrogenase reductase (20,31,32
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