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)

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.
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PMID:Demonstration and partial characterization of ADP-ribosylation in Pseudomonas maltophilia. 250 52

Treatment of fragment A chain of diphtheria toxin (DT-A) with diethylpyrocarbonate modifies His-21, the single histidine residue present in the chain, without alteration of other residues. Parallel to histidine modification, NAD+ binding and the NAD-glycohydrolase and ADP-ribosyltransferase activities of DT-A are lost. Both NAD+ and adenosine are very effective in protecting DT-A from histidine modification and in preserving its biological properties, while adenine is ineffective. Reversal of histidine modification with hydroxylamine restores both NAD+ binding and enzymatic activities of the toxin. The possible role of His-21 in the activity of diphtheria toxin is discussed in relation to the available three-dimensional structure of the related toxin produced by Pseudomonas aeruginosa.
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PMID:Histidine 21 is at the NAD+ binding site of diphtheria toxin. 252 25

Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist.
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PMID:Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli. 267 Sep

Directed mutagenesis was used to probe the functions of Tyr-470 and Tyr-481 of Pseudomonas aeruginosa exotoxin A (ETA) with respect to cytotoxicity, ADP-ribosylation of elongation factor 2 (EF-2), and NAD-glycohydrolase activity. Both of these residues lie in the active site cleft, close to Glu-553, a residue believed to play a direct role in catalysis of ADP-ribosylation of EF-2. Substitution of Tyr-470 with Phe caused no change in any of these activities, thus eliminating the possibility that the phenolic hydroxyl group of Tyr-470 might be directly involved in catalysis. Mutation of Tyr-481 to Phe caused an approximately 10-fold reduction in NAD:EF-2 ADP-ribosyltransferase activity and cytotoxicity but no change in NAD-glycohydrolase activity. The latter mutation did not alter the KM of NAD in the NAD-glycohydrolase reaction, which suggests that the phenolic hydroxyl of Tyr-481 does not participate in NAD binding. We hypothesize that the phenolic hydroxyl of Tyr-481 may be involved in the interaction of the toxin with substrate EF-2.
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PMID:Pseudomonas aeruginosa exotoxin A: effects of mutating tyrosine-470 and tyrosine-481 to phenylalanine. 284 95

The toxicity of pertussis toxin is mediated by the ADP-ribosyltransferase activity of subunit S1. To understand the structure-function relationship of subunit S1 and guide the construction of nontoxic molecules suitable for vaccines, we constructed and expressed in Escherichia coli a series of amino-terminal and carboxyl-terminal deletion mutants as well as a number of molecules containing amino acid substitutions. The shortest peptide still retaining enzymatic activity contains amino acids 2-179. Within this region we identified three mutants in which amino acid substitutions abolish the enzymatic activity. Mutation of amino acids 8 and 9 or 50 and 53, located within the region of the S1 subunit of pertussis toxin homologous to cholera toxin, causes loss of enzymatic activity. Outside this homology region, substitution of Glu-129 with glycine or aspartic acid also eliminates the enzymatic activity of the S1 subunit. In this respect, Glu-129 resembles the glutamic acid that is crucial for the catalytic activity of diphtheria and Pseudomonas toxins. Once introduced into the Bordetella pertussis chromosome, the above mutations should lead to the synthesis of nontoxic pertussis toxin molecules suitable for vaccine production.
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PMID:Subunit S1 of pertussis toxin: mapping of the regions essential for ADP-ribosyltransferase activity. 290 32

Exoenzyme S is an extracellular ADP-ribosyltransferase enzyme produced by Pseudomonas aeruginosa. Mutants of Pseudomonas aeruginosa deficient in this enzyme have been shown to have reduced virulence in infections of burned mice. The contribution of exoenzyme S to the pathogenesis of chronic lung infections with this organism was evaluated by examining the incidence of exoenzyme S production by Pseudomonas aeruginosa strains isolated from cystic fibrosis patients and comparing an exoenzyme S deficient mutant and its exoenzyme S producing parent in a rat chronic lung infection model. Of 51 isolates examined, 43% produced detectable levels of exoenzyme S. While both the exoenzyme S deficient mutant and its parent strain were equally capable of colonizing and persisting in rat lungs, the exoenzyme S producing parent elicited a greater degree of lung damage. These data suggest that exoenzyme S contributes to the pathogenesis of chronic lung infections.
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PMID:Role of exoenzyme S in chronic Pseudomonas aeruginosa lung infections. 298 45

Exotoxin A of Pseudomonas aeruginosa is a secreted bacterial toxin capable of translocating a catalytic domain into mammalian cells and inhibiting protein synthesis by the ADP-ribosylation of cellular elongation factor 2. The protein is a single polypeptide chain of 613 amino acids. The x-ray crystallographic structure of exotoxin A, determined to 3.0-A resolution, shows the following: an amino-terminal domain, composed primarily of antiparallel beta-structure and comprising approximately half of the molecule; a middle domain composed of alpha-helices; and a carboxyl-terminal domain comprising approximately one-third of the molecule. The carboxyl-terminal domain is the ADP-ribosyltransferase of the toxin. The other two domains are presumably involved in cell receptor binding and membrane translocation.
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PMID:Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. 300 45

The exotoxin A gene from Pseudomonas aeruginosa PAK was expressed in Escherichia coli from recombinant plasmids when transcription was initiated from a promoter in the cloning vector. The exotoxin A polypeptide synthesized was found to have an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of 66,000 daltons, identical in size to the mature exotoxin A made by P. aeruginosa. Analysis of the location of exotoxin A in various bacterial compartments by immunoblotting revealed that exotoxin A was exported by E. coli into its periplasmic space. Several functional assays, including analyses of disulfide bond formation, potentiation of ADP-ribosyltransferase activity, and HeLa cell cytotoxicity, were used to establish that the conformation of exotoxin A isolated from the E. coli periplasmic space is identical to that of exotoxin exported by P. aeruginosa to its extracellular space. Previous studies with recombinant plasmids expressing exotoxin A from P. aeruginosa PA103 (G. D. Gray, D. Smith, J. Baldridge, R. Markins, M. Vasil, E. Chen, and M. Heyneker, Proc. Natl. Acad. Sci. USA 81:2645-2649, 1984) showed a complete lack of processing and export of pre-exotoxin A in E. coli, differing from results reported here. These discrepancies may be explained by observed differences in the sequence of signal peptides encoded by the exotoxin A genes of PAK and PA103 strains of P. aeruginosa.
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PMID:Expression and secretion of the cloned Pseudomonas aeruginosa exotoxin A by Escherichia coli. 312 63

The exotoxin A genes from Pseudomonas aeruginosa strains PA103 and PAO1 have been independently cloned in a pUC9-derived plasmid. In a non-toxigenic mutant of PAO1 as host, the cloned genes directed the synthesis of intact exotoxin A that expressed ADP-ribosyltransferase activity upon treatment with urea and dithiothreitol. Western-blot analysis of culture supernatants identified a polypeptide of 67 kDa, the molecular mass of intact exotoxin A. There was an approximately 15-fold increase in the toxin yield from P. aeruginosa cells carrying a cloned PA103 gene compared to PA103, and a 40-fold increase in the yield of toxin gene yielded about four times more toxin than those carrying the cloned PAO1 gene. Toxin expression was correlated with the presence of a transcript that was initiated 88 bp upstream from the translational start site. Little or no messenger RNA from either cloned gene could be detected in an Escherichia coli host, or in a P. aeruginosa host grown in the presence of 0.1 mM-Fe2+, a condition that inhibits toxin expression. The nucleotide sequences of two regions, each of approximately 500 bp, near the 5' and 3' termini of the structural gene were established. In these regions, three exotoxin A gene from PAO1 has ten base-pair differences compared to the PA103 gene, three in the non-coding region, and seven in the structural gene, four of which should lead to amino-acid differences. No apparent sequence similarities were found between the inferred promoter region of the exotoxin A gene and that of other Pseudomonas genes, nor with the consensus sequence of E. coli promoters.
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PMID:Transcription and expression of the exotoxin A gene of Pseudomonas aeruginosa. 312 36

Pseudomonas aeruginosa exotoxin A (ETA) is inactivated greater than 1,000-fold when an active site glutamic acid, E553, is mutated to aspartic acid (Douglas, C.M., and Collier, R. J. (1987) J. Bacteriol. 169, 4967-4971). To test the effect of creating a carboxyl-containing side chain at position 553 longer than that of glutamic acid, we first replaced Glu-553 with cysteine by site-directed mutagenesis of cloned ETA and then carboxymethylated the cysteine side chain with iodoacetic acid. The E553C mutation reduced ADP-ribosyltransferase and cytotoxic activities greater than 10,000-fold. Reaction of the mutant with iodoacetic acid enhanced enzymic activity 2,500-fold, to a level approximately one-sixth that of wild type toxin, and restored cytotoxicity to a slightly lesser extent. Iodoacetamide did not activate the mutant, and neither iodoacetic acid nor iodoacetamide affected the activity of wild type toxin. These results show that the carboxyl group of Glu-553 is important for ADP-ribosylation activity and imply flexibility in the enzyme-substrate complex in accommodating the slightly longer S-carboxymethylcysteine side chain. This general approach may have applications in protein engineering as well as in studying carboxyl side chain functions in enzymes.
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PMID:Restoration of enzymic activity and cytotoxicity of mutant, E553C, Pseudomonas aeruginosa exotoxin A by reaction with iodoacetic acid. 312 20


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