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:4.1.2.42 (DTA)
1,693 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A segment of the exotoxin A gene of Pseudomonas aeruginosa, coding for the N-terminal end of domain I and domain II of the toxin (ETA), was genetically fused to the diphtheria toxin gene of Corynebacterium diphtheriae, coding for the N-terminal end of A fragment of diphtheria toxin (DT). The resulting hybrid protein (termed CED1) was produced in large amounts and exported to the periplasm in Escherichia coli. This chimaeric protein reacted with both anti-ETA and anti-DT antisera. Furthermore, the chimaeric protein displayed ADP-ribosylation activity and exhibited cytotoxicity to mouse 3T6 fibroblasts. These results demonstrated that the chimaeric protein is cytotoxic, and that the toxic potential of DTA can be selectively internalized and translocated via domains I and II of exotoxin A, which are thus sufficient to direct and translocate an enzymatically active heterologous polypeptide segment into the cytosol of sensitive cells.
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PMID:Cytotoxic activity of a recombinant chimaeric protein between Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin. 164 Aug 30

Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. 198 Feb 8

The role of discrete domains of diphtheria toxin (DT) B chain in cytosol entry and cytotoxicity was investigated by linking a monoclonal antibody recognizing the human T cell-specific antigen T3 (UCHT1) to diphtheria toxin (UCHT1-DT), DT A subunit (UCHT1-DTA), or to a genetically engineered form of DT (UCHT1-MspSA) lacking the C-terminal 17-kDa portion of the B subunit. The N-terminal 21-kDa region of DT B chain increased toxicity of UCHT1-DTA 100-fold (UCHT1-MspSA) while addition of the C-terminal 17-kDa region (UCHT1-DT) increased toxicity 100-fold more. The cytotoxicity was dependent upon antibody binding as demonstrated by blocking toxicity with excess UCHT1. The differences in toxicity between these reagents were not due to differences in ADP-ribosylation activity of DT A chain, binding activity of the antibody moiety, extent of DT nicking, or the cross-linking method, so we conclude that the large differences in toxicity were due to the presence of different B chain domains. The large increase in toxicity by the C-terminal region of DT B did not appear to be caused by DT receptor binding. The lysosomotropic agent NH4Cl blocked the cytotoxic effect of DT, UCHT1-DT, and UCHT1-MspSA but not UCHT1-DTA.
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PMID:Cloned fragment of diphtheria toxin linked to T cell-specific antibody identifies regions of B chain active in cell entry. 348 93

Previous studies have suggested that tyrosine-65 (Tyr-65) of diphtheria toxin (DT) is located at the active site. To investigate the role of Tyr-65 in NAD binding and the ADP-ribosylation of elongation factor-2 (EF-2), we changed this residue to alanine and phenylalanine by site-directed mutagenesis of a synthetic gene encoding the catalytic fragment of DT (DTA). The alanine mutant was greatly diminished in ADP-ribosylation activity (350-fold) and NAD-glycohydrolase activity (88-fold), whereas the phenylalanine mutant was reduced in these activities only slightly. Dissociation constants (Kd) for NAD binding were 15 microM for wild-type DTA, 26 microM for the phenylalanine mutant, and greater than 800 microM NAD for the alanine mutant. However, both mutant enzymes were found to bind adenosine with nearly equal affinity as wild-type DTA. These results support a model of ADP-ribosylation in which the phenolic ring of Tyr-65 interacts with the nicotinamide ring of NAD, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism.
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PMID:Active-site mutations of diphtheria toxin: role of tyrosine-65 in NAD binding and ADP-ribosylation. 780 11

Diphtheria toxin (DT) has been studied as a model for understanding active-site structure and function in the ADP-ribosyltransferases. Earlier evidence suggested that histidine-21 of DT is important for the ADP-ribosylation of eukaryotic elongation factor 2 (EF-2). We have generated substitutions of this residue by cassette mutagenesis of a synthetic gene encoding the catalytic A fragment (DTA) of DT, and have characterized purified mutant forms of this domain. Changing histidine-21 to alanine, aspartic acid, leucine, glutamine, or arginine diminished ADP-ribosylation activity by 70-fold or greater. In contrast, asparagine proved to be a functionally conservative substitution, which reduced ADP-ribosylation activity by < 3-fold. The asparagine mutant was approximately 50-fold-attenuated in NAD glycohydrolase activity, however. Dissociation constants (Kd) for NAD binding, determined by quenching of the intrinsic protein fluorescence, were 15 microM for wild-type DTA, 160 microM for the asparagine mutant, and greater than 500 microM NAD for the alanine, leucine, glutamine, and arginine mutants. These and previous results support a model of the ADP-ribosylation of EF-2 in which histidine-21 serves primarily a hydrogen-bonding function. We propose that the pi-imidazole nitrogen of His-21 hydrogen-bonds to the nicotinamide carboxamide, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism, and then stabilizing the transition-state intermediate of this reaction.
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PMID:Active-site mutations of the diphtheria toxin catalytic domain: role of histidine-21 in nicotinamide adenine dinucleotide binding and ADP-ribosylation of elongation factor 2. 817 90

CRM45 is a mutant form of diphtheria toxin (DTx) that lacks a 17-kDa carboxyl-terminal segment of the receptor-binding B subunit (DTB). The missing segment is a discrete structural domain of DTB that normally rests against the NAD binding pocket of the enzymically-active A subunit (DTA). Proteolytic cleavage and disulfide bridge reduction in the DTA-DTB linker region of DTx are required for optimal ADP-ribosylation of elongation factor 2 (EF-2). Here, we show that cleaved and uncleaved preparations of X-ray crystal grade CRM45 both exhibit an ADP-ribosyltransferase activity similar to that of cleaved DTx. Crystal-grade preparations of CRM45 also display a potent deoxyribonuclease activity. However, as observed with DTx, cleavage and reduction of CRM45 are not required for expression of this nuclease activity. After SDS-PAGE in a gel that contains DNA embedded in the matrix, renaturable Ca++/Mg(++)-dependent nuclease-active bands co-migrate with intact CRM45 (45 kDa) as well as with the DTA subunit (24 kDa) of CRM45. Because the 45-kDa nuclease-active band is unique to the CRM45 form of DTx, it offers direct proof that this activity is intrinsic to the DTA domain of DTx and its homologues.
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PMID:Characterization of the deoxyribonuclease and ADP-ribosyltransferase activities of CRM45, a truncated homologue of diphtheria toxin. 978 63

The bacterial exotoxins, cholera toxin (CT), pertussis toxin (PT), and diphtheria toxin (DT), interfere with specific host proteins to cause tissue damage for their respective infections. The common toxic mechanism for these agents is mono-ADP-ribosylation of specific amino acids in G(s)(alpha), G(i)(alpha), and eEF-2 proteins, respectively, by the catalytic A chains of the toxins (CTA, PTA, and DTA). In the absence of acceptor proteins, these toxins also act as NAD(+)-N-ribosyl hydrolases. The transition-state structures for NAD(+) hydrolysis and ADP-ribosylation reactions have oxacarbenium ion character in the ribose. We designed and synthesized analogues of NAD(+) to resemble their oxacarbenium ion transition states. Inhibitors with oxacarbenium mimics replacing the NMN-ribosyl group of NAD(+) show 200-620-fold increased affinity in the hydrolytic and N-ribosyl transferase reactions catalyzed by CTA. These analogues are also inhibitors for the hydrolysis of NAD(+) by PTA with K(i) values of 24-40 microM, but bind with similar affinity to the NAD(+) substrates. Inhibition of the NAD(+) hydrolysis and ADP-ribosyl transferase reactions of DTA gave K(i) values from 19 to 48 microM. Catalytic rate enhancements by the bacterial exotoxins are small, and thus transition-state analogues cannot capture large energies of activation. In the cases of DTA and PTA, analogues known to resemble the transition states bind with approximately the same affinity as substrates. Transition-state analogue interrogation of the bacterial toxins indicates that CTA gains catalytic efficiency from modest transition-state stabilization, but DTA and PTA catalyze ADP-ribosyl transferase reactions more from ground-state destabilization. pH dependence of inhibitor action indicated that both neutral and cationic forms of transition-state analogues bind to DTA with similar affinity. The origin of this similarity is proposed to reside in the cationic nature of NAD(+) both as substrate and at the transition state.
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PMID:Inhibitors of ADP-ribosylating bacterial toxins based on oxacarbenium ion character at their transition states. 1512 61

Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LF(N)DTA. LF(N) mediates uptake of LF(N)DTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP-ribosylates elongation factor-2, allowing for detection of small amounts of translocated LF(N)DTA. Cyclosporin A, which inhibits peptidyl-prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LF(N)DTA into the cytosol of CHO-K1 cells and protected cells from intoxication by LF(N)DTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LF(N)DTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LF(N)DTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LF(N)DTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.
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PMID:Role of CypA and Hsp90 in membrane translocation mediated by anthrax protective antigen. 2094 44

2-oxo-4-phenyl-6-styryl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid (ADP) was complexed with acetates of Mn(II), Ni(II), Cu(II) and Zn(II). The structures of the ligand and its metal complexes were characterized by microanalysis, IR, NMR, UV-vis spectroscopy, magnetic susceptibility and TGA-DTA analyses. Octahedral and square planar geometries were suggested for the complexes in which the central metal ion coordinated with O donors of ligand and acetate ions. Each ligand binds the metal using carboxylate oxygens. The ligand and complexes were evaluated for their antimicrobial activities against different species of pathogenic bacteria and fungi. The present novel pyrimidine containing complexes could constitute a new group of antibacterial and antifungal agents.
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PMID:Spectral and in vitro antimicrobial properties of 2-oxo-4-phenyl-6-styryl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid transition metal complexes. 2248 5

Diphtheria toxin is a single-chain protein toxin that invades human cells by receptor-mediated endocytosis. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA) from endosomal lumen into the host cell cytosol. Here, DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death. The compound 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA) has been previously shown to protect cells from various bacterial protein toxins which deliver their enzymatic subunits from acidic endosomes to the cytosol, including Bacillus anthracis lethal toxin and the binary clostridial actin ADP-ribosylating toxins C2, iota and Clostridium difficile binary toxin (CDT). Here, we demonstrate that EGA also protects human cells from diphtheria toxin by inhibiting the pH-dependent translocation of DTA across cell membranes. The results suggest that EGA might serve for treatment and/or prevention of the severe disease diphtheria.
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PMID:Semicarbazone EGA Inhibits Uptake of Diphtheria Toxin into Human Cells and Protects Cells from Intoxication. 2742 99


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