Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enzyme that catalyzes the ADP-ribosylation and concomitant inactivation of dinitrogenase reductase in Rhodospirillum rubrum has been purified greater than 19,000-fold to near homogeneity. We propose dinitrogenase reductase ADP-ribosyltransferase (DRAT) as the working name for the enzyme. DRAT activity is stabilized by NaCl and ADP. The enzyme is a monomer with a molecular mass of 30 kDa and is a different polypeptide than dinitrogenase reductase activating glycohydrolase. NAD (Km = 2 mM), etheno-NAD, nicotinamide hypoxanthine dinucleotide, and nicotinamide guanine dinucleotide will serve as donor molecules in DRAT-catalyzed ADP-ribosylation reaction, and dinitrogenase reductases from R. rubrum, Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianium will serve as acceptors. No other proteins or small molecules, including water, have been found to be effective as acceptors. Nicotinamide is released stoichiometrically with formation of the ADP-ribosylated product. DRAT is inhibited by NaCl and has maximal activity at a pH of 7.0.
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PMID:Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum. 314 11

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation.
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PMID:Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase. 783 96

Several cases of ADP-ribosylation of endogenous proteins in procaryotes have been discovered and investigated. The most thoroughly studied example is the reversible ADP-ribosylation of the dinitrogenase reductase from the photosynthetic bacterium Rhodospirillum rubrum and related bacteria. A dinitrogenase reductase ADP-ribosyltransferase (DRAT) and a dinitrogenase reductase ADP-ribose glycohydrolase (DRAG) from R. rubrum have been isolated and characterized. The genes for these proteins have been isolated and sequences and show little similarity to the ADP-ribosylating toxins. Other targets for endogenous ADP-ribosylation by procaryotes include glutamine synthetase in R. rubrum and Rhizobium meliloti and undefined proteins in Streptomyces griseus and Pseudomonas maltophila.
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PMID:Reversible ADP-ribosylation as a mechanism of enzyme regulation in procaryotes. 789 54

From Azospirillum lipoferum (Al) FS, a nitrogen-fixing bacterium isolated from the rhizosphere of rice, we cloned and sequenced draT, encoding dinitrogenase reductase ADP-ribosyltransferase, and draG, encoding dinitrogenase reductase-activating glycohydrolase. The nucleotide sequences of draTG showed extensive similarity to the same genes from Azospirillum brasilense, Rhodospirillum rubrum and Rhodobacter capsulatus, and they are assumed to be co-transcribed as a single operon. When this draTG operon was introduced into Klebsiella oxytoca, this organism acquired the ability to respond to extracellular NH(+4) ions with reversible inhibition of nitrogenase activity, similar to that seen in Al FS. We constructed a plasmid containing a draT::lacZ gene fusion and found that beta-galactosidase activity was detected under microaerobic conditions, regardless of NH(+4) concentration, but not under aerobic conditions. This indicates that the transcription of draTG responds to the level of oxygen, but not to that of NH(+4) ions.
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PMID:Cloning, sequencing and transcriptional regulation of the draT and draG genes of Azospirillum lipoferum FS. 862 Oct 68

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.
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PMID:Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium. 863 86

A key step in the action of cholera toxin (CT) is the reduction of its A subunit to the A1 peptide. The latter is an ADP-ribosyltransferase, which activates the alpha-subunit of the stimulatory G protein of adenylyl cyclase. In this study, the enzymatic reduction of membrane-bound CT in CaCo-2 human intestinal epithelial cells was characterized. Whereas diphtheria toxin was found to be reduced by a cell surface population of protein-disulfide isomerase (PDI) and its cytotoxicity was inhibited by p-chloromercuribenzenesulfonic acid, bacitracin, or anti-PDI antibodies, these inhibitors had no effect on CT reduction or activity in intact cells. In contrast, the reduction of CT in vitro by either postnuclear supernatants (PNS) or microsomal membranes in the presence of Triton X-100 was significantly inhibited by p-chloromercuribenzenesulfonic acid and bacitracin. Anti-PDI monoclonal antibodies likewise inhibited the in vitro reduction of CT and also were effective in depleting reductase activity from PNS. Since inhibition and depletion were not observed in the absence of detergent, these results suggested that the reductase activity was a soluble component localized to the lumen of microsomal vesicles and correlated with the presence of protein-disulfide isomerase. This was further confirmed by showing a corresponding depletion of reductase activity and PDI in alkali-treated microsomes. This activity was restored when purified bovine PDI was added back to alkali-treated microsomes in a redox buffer that reflected conditions found in the lumen of the endoplasmic reticulum (ER). When the CT-related reductase activity was assayed in subcellular fractions of PNS-derived membranes isolated on a 9-30% Iodixanol gradient, the activity, as measured by CT-A1 peptide formation localized to those fractions containing PDI. Likewise CT-A1 peptide formed in intact cells co-localized to those membrane fractions containing the majority of cellular PDI. Furthermore, the banding density corresponded to a region of the gradient containing ER-derived membranes. These results indicated that CT was a substrate for PDI-catalyzed reduction in intact cells and supported the hypothesis that CT reduction and activation occurs in the ER.
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PMID:Protein-disulfide isomerase-mediated reduction of the A subunit of cholera toxin in a human intestinal cell line. 902 Jan 87

The role of NAD+ in the metabolic regulation of nitrogenase, the 'switch-off' effect, in Rhodospirillum rubrum has been studied. We now show that the decrease in nitrogenase activity upon addition of NAD+ to R. rubrum is due to modification of dinitrogenase reductase. There was no effect when NAD+ was added to a mutant of R. rubrum devoid of dinitrogenase reductase ADP-ribosyltransferase, indicating that NAD+ 'switch-off' is an effect of the same regulatory system as ammonium 'switch-off'. We also show that oxaloacetate and alpha-ketoglutarate function as 'switch-off' effectors. On the other hand beta-hydroxybutyrate has the opposite effect by shortening the 'switch-off' period. Furthermore, by using an inhibitor of glutamate synthase the role of this enzyme in 'switch-off' was investigated. The results are discussed in relation to our proposal that changes in the concentration of NAD+ are involved in initiating 'switch-off'.
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PMID:The role of NAD+ as a signal during nitrogenase switch-off in Rhodospirillum rubrum. 914 56

Chemical cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum has been investigated with a cross-linking system utilizing two reagents, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and sulfo-N-hydroxysuccinimide. Cross-linking between dinitrogenase reductase and DRAT requires the presence of NAD, the cellular ADP-ribose donor, or a NAD analog containing an unmodified nicotinamide group, such as nicotinamide hypoxanthine dinucleotide. NADP, which will not replace NAD in the modification reaction, does support cross-linking between dinitrogenase reductase and DRAT. The DRAT-catalyzed ADP-ribosylation of dinitrogenase reductase is inhibited by sodium chloride, as is the cross-linking between dinitrogenase reductase and DRAT, suggesting that ionic interactions are required for the association of these two proteins. Cross-linking is specific for native, unmodified dinitrogenase reductase, in that both oxygen-denatured and ADP-ribosylated dinitrogenase reductase fail to form a cross-linked complex with DRAT. The ADP-bound and adenine nucleotide-free states of dinitrogenase reductase form cross-linked complexes with DRAT; however, cross-linking is inhibited when dinitrogenase reductase is in its ATP-bound state.
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PMID:NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum. 915 Feb 24

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.
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PMID:Correlation of activity regulation and substrate recognition of the ADP-ribosyltransferase that regulates nitrogenase activity in Rhodospirillum rubrum. 1004 7

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif 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 of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-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 NH(4)(+) and darkness treatments.
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PMID:Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum. 1064 24


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