The genetics and biochemistry of the reversible ADP-ribosylation systems of Rhodospirillum rubrum and Azospirillum lipoferum

1990 ◽  
pp. 475-481 ◽  
Author(s):  
G. P. Roberts ◽  
P. W. Ludden ◽  
R. H. Burris ◽  
W. P. Fitzmaurice ◽  
H.-A. Fu ◽  
...  
Keyword(s):  
Rhodospirillum Rubrum ◽  
Azospirillum Lipoferum ◽  
Adp Ribosylation

scholarly journals Correlation of Activity Regulation and Substrate Recognition of the ADP-Ribosyltransferase That Regulates Nitrogenase Activity in Rhodospirillum rubrum

1999 ◽  
Vol 181 (5) ◽  
pp. 1698-1702 ◽  
Author(s):  
Kitai Kim ◽  
Yaoping Zhang ◽  
Gary P. Roberts
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Substrate Recognition ◽  
Posttranslational Regulation ◽  
Activity Regulation ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT In Rhodospirillum rubrum, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected.

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

scholarly journals Demonstration and partial characterization of ADP-ribosylation in Pseudomonas maltophilia

1989 ◽  
Vol 261 (1) ◽  
pp. 113-118 ◽  
Author(s):  
C Edmonds ◽  
G E Griffin ◽  
A P Johnstone
Keyword(s):  
Rhodospirillum Rubrum ◽  
Gel Filtration ◽  
Protein S ◽  
Exchange Chromatography ◽  
Separate Species ◽  
Adp Ribosylation ◽  
Nad Glycohydrolase ◽  
Substrate Protein ◽  
Pseudomonas Maltophilia ◽  
Adp Ribosyltransferase

ADP-ribosylation of proteins occurs in many eukaryotes, and it is also the mechanism of action of a growing number of important bacterial toxins. To date, however, there is only one well-characterized ADP-ribosylation system where the ADP-ribosyltransferase and the substrate protein are both bacterial in origin, namely within the nitrogen-fixing bacterium Rhodospirillum rubrum. The present paper demonstrates the endogenous ADP-ribosylation of two proteins of Mr 32,000 and 20,000 within Pseudomonas maltophilia, a Gram-negative aerobe. The proteins have been partially purified: two apparently separate species of modified protein can be separated by ion-exchange chromatography and gel filtration (V0 and Mr 158,000 - Vi). The substrate protein(s) either has, or is co-eluted with, NAD+ glycohydrolase activity. The modification is mono-ADP-ribosyl in nature. The linkage between the acceptor amino acid and the ADP-ribose moiety is alkali-labile and stable to hydroxylamine, possibly indicating an S-glycosidic bond. The activity appears to be a true ADP-ribosylation reaction and not an NAD+ glycohydrolase activity followed by non-enzymic addition of ADP-ribose to protein. The results presented here indicate that ADP-ribosylation may have a wider significance within prokaryotic systems than previously thought.

Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum

1989 ◽  
Vol 218 (2) ◽  
pp. 340-347 ◽  
Author(s):  
Wayne P. Fitzmaurice ◽  
Leonard L. Saari ◽  
Robert G. Lowery ◽  
Paul W. Ludden ◽  
Gary P. Roberts
Keyword(s):  
Rhodospirillum Rubrum ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase

scholarly journals Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation.

1991 ◽  
Vol 173 (21) ◽  
pp. 6903-6909 ◽  
Author(s):  
J H Liang ◽  
G M Nielsen ◽  
D P Lies ◽  
R H Burris ◽  
G P Roberts ◽  
...  
Keyword(s):  
Rhodospirillum Rubrum ◽  
Adp Ribosylation

scholarly journals Mutagenesis and Functional Characterization of theglnB, glnA, and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

2000 ◽  
Vol 182 (4) ◽  
pp. 983-992 ◽  
Author(s):  
Yaoping Zhang ◽  
Edward L. Pohlmann ◽  
Paul W. Ludden ◽  
Gary P. Roberts
Keyword(s):  
Nitrogen Fixation ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Functional Characterization ◽  
Photosynthetic Bacterium ◽  
Wild Type ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation ofnif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase–dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization ofglnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for PII) were constructed. While PII-Y51F showed a lower nitrogenase activity than that of wild type, a PIIdeletion mutant showed very little nif expression. This effect of PII on nif expression is apparently the result of a requirement of PII for NifA activation, whose activity is regulated by NH4 + in R. rubrum. The modification of glutamine synthetase (GS) in theseglnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of PII might exist inR. rubrum and regulate the modification of GS. PII also appears to be involved in the regulation of DRAT activity, since an altered response to NH4 + was found in a mutant expressing PII-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH4 + and darkness treatments.

scholarly journals Effect of PII and Its Homolog GlnK on Reversible ADP-Ribosylation of Dinitrogenase Reductase by Heterologous Expression of the Rhodospirillum rubrum Dinitrogenase Reductase ADP-Ribosyl Transferase–Dinitrogenase Reductase-Activating Glycohydrolase Regulatory System inKlebsiella pneumoniae

2001 ◽  
Vol 183 (5) ◽  
pp. 1610-1620 ◽  
Author(s):  
Yaoping Zhang ◽  
Edward L. Pohlmann ◽  
Cale M. Halbleib ◽  
Paul W. Ludden ◽  
Gary P. Roberts
Keyword(s):  
Klebsiella Pneumoniae ◽  
Heterologous Expression ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Expression System ◽  
Regulatory System ◽  
Wild Type ◽  
Heterologous Expression System ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase

ABSTRACT Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase–dinitrogenase reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnBmutant, with DRAG being inactivated more slowly in response to NH4 + treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In aglnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH4 + addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation inR. rubrum.

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