Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine

Wilson, B A; Reich, Karl; Weinstein, B R; Collier, R. J.

doi:10.1021/bi00489a021

Cited by 96 publications

(105 citation statements)

References 35 publications

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“…Although the amino acid sequence homology among various ADPribosyltransferases is poor, there is a highly conserved glutamic acid residue, like the Glu-112 of the cholera toxin A subunit and the Glu-148 of diphtheria toxin (16,23,33). A number of mutagenesis assays have indicated that such glutamic acid residues are essential for exertion of toxicity and ADPribosylating activity (34)(35)(36)(37)(38). X-ray crystallographic and photocrosslinking analyses suggest that the glutamic acid residue serves as an NAD-binding site (21,39,40).…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: Possible involvement of ADP-ribosylation in its activity

Watanabe

Kono

Matsushima-Hibiya

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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We have previously reported that the cabbage butterf ly, Pieris rapae, contains a 98-kDa protein, named pierisin, that induces apoptosis in a variety of human cancer cell lines. In the present study, sequencing and cloning of a cDNA encoding pierisin was accomplished. PCR-direct sequencing showed that the gene encodes an 850-amino acid protein with a calculated molecular weight of 98,081. An intact clone at the amino acid level encompassing the entire coding region was obtained by recombination of two independent clones, and the molecular mass of its in vitro expressed protein was about 100 kDa on SDS͞PAGE, the same as that of purified native pierisin. The expressed protein induced apoptosis in human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, like the native protein, indicating functional activity. The deduced amino acid sequence of pierisin showed 32% homology with a 100-kDa mosquitocidal toxin from Bacillus sphaericus SSII-1. In addition, pierisin showed regional sequence similarities with ADP-ribosylating toxins, such as the A subunit of cholera toxin. A glutamic acid residue at the putative NAD-binding site, conserved in all ADPribosylating toxins, was also found in pierisin. Substitution of another amino acid for glutamic acid 165 resulted in a great decrease in cytotoxicity and induction of apoptosis. Moreover, inhibitors of ADP-ribosylating enzymes reduced pierisininduced apoptosis. These results suggest that the apoptosisinducing protein pierisin might possess ADP-ribosylation activity that leads to apoptosis of the cells.

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Section: Discussionmentioning

confidence: 99%

Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: Possible involvement of ADP-ribosylation in its activity

Watanabe

Kono

Matsushima-Hibiya

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…In other studies, we have constructed a series of GALl promoter expression plasmids that contain the gene for the A fragment of DT and several mutant derivatives of the activesite residue, Glu-148 (17). Two of these mutants, containing changes of Glu-148 to Asp and Glu-148 to Ser, have reductions in NAD:EF-2 ADP-ribosyltransferase activity of 100-and 300-fold, respectively (50), while the third mutant, containing a deletion of Glu-148, has approximately times the activity of wild-type A fragment (22 4 and 6. Densitometric scanning of the autoradiogram showed that the intensity of the 100-kDa EF-2 band from lanes 1 to 3 correlated with toxin concentration.…”

Section: Scdm5 26 C S R E]y L E H Y T J-i Lj]a a S Q E El E S Y 49mentioning

confidence: 99%

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

Mattheakis¹,

Shen²,

Collier³

1992

Mol. Cell. Biol.

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A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPHS. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen Il methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen Ill methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dphS mutant. A null mutation of the chromosomal DPHS gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fuly active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.

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“…Thus, it is possible that the nicotinamide ring stacks against Tyr 65 in a similar fashion in DT-NAD. Glu 148 is the only residue in DT that was previously shown to play a role in catalysis; its mutation even to the chemically similar residues Gln or Asp greatly diminishes ADP-ribosylation activity (Wilson et al, 1990). Therefore, Glu 148 is expected to be near the active site.…”

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confidence: 99%

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

1994

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The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections ( F > l o ( F ) ) , yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp.The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.Keywords: ADP-ribosyltransferase; diphtheria; membrane insertion Diphtheria toxin (DT) is secreted from toxic strains of the bacterium Corynebacterium diphtheriae. Toxigenic conversion of C. diphtheriae occurs by lysogenization with corynephage 0 (Freeman, 1951), which carries the DT gene encoding a 535-residue protein (M, 58,342) (Uchida et al., 1971; Greenfield et al., 1983). DT kills eukaryotic cells by inactivating an essential component of the protein translation machinery, elongation factor 2 (EF-2) (Collier, 1975).DT is produced as a protomer of 2 fragments connected by a loop containing a proteolysis site and a disulfide bond. Limited proteolysis with trypsin and reduction of the disulfide separates the 2 fragments, which are denoted fragment A and fragment B (Drazin et al., 1971). Fragment A consists of an independent folding domain, the catalytic (C) domain (residues 1-190) (Choe et al., 1992). Fragment B consists of 2 folding domains, the transmembrane (T) domain (residues 191-378) and receptor-binding (R) domain (residues 379-535) (Choe et al., 1992).

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Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine

Cited by 96 publications

References 35 publications

Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: Possible involvement of ADP-ribosylation in its activity

Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: Possible involvement of ADP-ribosylation in its activity

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

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