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Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Botulism is widely known to result from ingestion of food containing botulinum neurotoxin produced in situ by certain strains of Clostridium botulinum. Infant botulism caused by C. botulinum, unlike the food-borne intoxication, is the toxicoinfectious form of botulism (S. S. Arnon, p. 331-345, in G. E. Lewis, ed., Biomedical Aspects of Botulism, 1981). The strain of Clostridium baratii implicated in infant botulism produced a neurotoxin that was neutralized with antiserum for botulinum neurotoxin serotype F (J. D. Hall, L. M. McCroskey, B. J. Pincomb, and C. L. Hatheway, J. Clin. Microbiol. 21:654-655, 1985). We developed a procedure to culture the toxigenic C. baratii (strain 6341) in dialysis bags and a simple purification scheme (precipitation of 900-ml culture supernatant with ammonium sulfate and two anion-exchange chromatographic steps at pH 5.5 and 8.0) that yielded up to 150 micrograms of purified neurotoxin. It is an approximately 140-kDa single-chain protein and has the following sequence of amino acid residues at the N terminus: Pro-Val-Asn-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asn-Asn-Thr-Thr-Ile- Leu. Comparison of this amino acid sequence with those of the botulinum neurotoxin serotypes A, B, and E showed 40 to 50% identical residues in comparable positions. The specific toxicity of the neurotoxin, approximately 2 x 10(6) 50% lethal doses for mice per mg of protein injected, was not enhanced significantly by mild trypsinization, although the protease cleaved the neurotoxin within a disulfide loop that generated at least two primary fragments, approximately 47 and approximately 86 kDa, that remained linked by an interchain disulfide. These two fragments resembled the light and heavy chains of the well-characterized neurotoxin serotypes A, B, C, D, E, and F produced by C. botulinum.
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PMID:Characterization of the neurotoxin isolated from a Clostridium baratii strain implicated in infant botulism. 173 Apr 84

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.
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PMID:Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site. 212 6

The 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall). The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond. The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT [1987) FEBS Lett. 226, 115-120). Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies. Two different approaches were taken to cut the H chain with trypsin and isolate the fragments. In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment. The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography. The 1-27 residues of 49-kDa N-terminal half of the H chain were Ala-Leu-Asn-Asp-Leu-Cys-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe-Phe-Ser-Pro- Ser-Glu - Asp-Asn-Phe-Thr-Asn-Asp-Leu-. The sequence of the other half of the H chain (44 kDa) was X-Ile-Ile-Asn-Leu-X-Ile-Leu-Asn-Leu-Arg-Tyr-Glu-X-Asn-His-Leu-Ile-Asp-Le u-Lys- X-Tyr-Ala-Ser-. In the second method, the H chain was first separated from the L chain, purified, and then cleaved. One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method. The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain. (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences. 317 18

A novel assay method based on the endopeptidase activities of the botulinum neurotoxins has been developed and applied to the detection of botulinum type A and B toxins. An assay system developed for the detection of botulinum type B neurotoxin (BoNT/B) is based on the cleavage of a synthetic peptide substrate representing amino acid residues 60 to 94 of the intracellular target protein for the toxin, VAMP (vesicle-associated membrane protein, or synaptobrevin). In this assay system, immobilized VAMP (60-94) peptide substrate is cleaved by BoNT/B at the Gln-76-Phe-77 bond, leaving the C-terminal cleavage fragment on the solid phase. This fragment is then detected by the addition of an antibody-enzyme reagent which specifically recognizes the newly exposed N terminus of the cleavage product. The developed assay was specific to BoNT/B, showing no cross-reactivity with other clostridial neurotoxins, and had a sensitivity for BoNT/B of 0.6 to 4.5 ng/ml, which could be increased to 0.1 to 0.2 ng/ml by using an assay amplification system based on catalyzed reporter deposition. Trypsin treatment of BoNT/B samples, which converts the single-chain toxin to the active di-chain form, was found to increase the sensitivity of the endopeptidase assay from 5- to 10-fold. An endopeptidase assay for BoNT/A, based on the cleavage of a peptide substrate derived from the protein SNAP-25 (synaptosome-associated protein), was also developed and characterized.
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PMID:Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities. 881 85

Zn2+-protease activity of botulinum neurotoxin causes the blockage of neurotransmitter release resulting in botulism disease. We have investigated the role of Zn2+ in the biological activity of type A botulinum neurotoxin by removing the bound Zn2+ by EDTA treatment, followed by monitoring its structure in terms of secondary and tertiary folding (second derivative UV, FT-IR, and circular dichroism spectroscopy) and function in terms of its effect on the release of norepinephrine from PC12 cells. The single Zn2+ bound to each neurotoxin molecule was reversibly removed by EDTA treatment, whereas the biological activity of the neurotoxin was irreversibly lost. Based on the Amide III IR spectral analysis, the alpha-helical content of neurotoxin increased from 29% to 42% upon removal of Zn2+, which reverted to 31% upon treatment with 1:5 molar excess of exogenous Zn2+. Second derivative UV spectroscopy revealed no change in surface topography of Tyr residues with removal of Zn2+. However, near-UV circular dichroism signals suggested significant alterations in the topography of Phe and Tyr residues that could be buried in the protein matrix. Thermal unfolding experiments suggested that removal of Zn2+ results in the formation of the molten globule-like structure of type A botulinum neurotoxin. Tertiary structural changes introduced by Zn2+ removal were irreversible, which correlated well with the irreversibility of the biological activity of the neurotoxin. On the basis of these results, we suggest that Zn2+ plays a significant structural role in addition to its catalytic role in Zn2+-protease activity of type A botulinum neurotoxin.
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PMID:Role of zinc in the structure and toxic activity of botulinum neurotoxin. 954 58

Three putative metalloprotease inhibitors were synthesized and tested for their ability to inhibit the catalytic activity of botulinum neurotoxin B light chain (BoNT/B LC). The compounds were designed to emulate the naturally occurring metalloprotease inhibitor phosphoramidon, which has been reported to be a weak antagonist of BoNT/B action. All three analogs contained the dipeptide Phe-Glu in place of Leu-Trp of phosphoramidon and possessed a phenyl, ethyl or methyl group in place of the rhamnose sugar of the parent compound. The inhibitors were evaluated in a cell-free assay based on the detection of a fluorescent product following cleavage of a 50-mer synaptobrevin peptide ([Pya(88)] S 39-88) by BoNT/B LC. This peptide corresponds to the hydrophilic core of synaptobrevin-2 and contains a fluorescent analog L-pyrenylalanine (Pya) in place of Tyr(88). Cleavage of [Pya(88)] S 39-88 by BoNT/B LC gives rise to fragments of 38 and 12 amino acid residues. Quantification of BoNT/B-mediated substrate cleavage was achieved by separating the 12-mer fragment (FETSAAKLKRK-Pya) that contains the C-terminal fluorophore and measuring fluorescence at 377 nm. The results indicate that the phenyl-substituted synthetic compound ICD 2821 was slightly more active than phosphoramidon, but analogs with methyl or ethyl substitutions were relatively inactive. These findings suggest that phosphonate monoesters may be useful for providing insights into the structural requirement of BoNT/B protease inhibitors.
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PMID:Evaluation of phosphoramidon and three synthetic phosphonates for inhibition of botulinum neurotoxin B catalytic activity. 1059 92

Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the amino acids glutamine and phenylalanine (Q and F bond) in position 76-77 of synaptobrevin (VAMP2). We evaluated peptides that contain the QF cleavage site but are not identical in primary structure to the VAMP2 sequence surrounding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B. A reverse-phase high-performance liquid chromatography (RP-HPLC) method was used to measure digested peptides. A dose as high as 600 microM of substance P, and 11-amino acid peptide containing the QF bond, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B. Buforin I (B-I, QF site 24-25) is 39 amino acids in length, and sequence comparison of B-I and VAMP2 indicated a similarity of 18% for conserved amino acids around the QF site. Furthermore, computer-aided secondary structure computations predict alpha-helical structures flanking the QF site for VAMP2 and for the upstream sequence of B-I. Although predictions for the downstream sequence give nearly equal tendencies for alpha-helical and beta-sheet structures, Yi et al. showed that the downstream sequence is likely to be the alpha-helix based on their examination of buforin II (B-II, a 21-amino acid subset of B-I (16-36)), which includes the QF site and the downstream sequence of B-I. Buforin I was found not to be a substrate for BoNT/B; however, B-I dose dependently and competitively inhibited BoNT/B activity, yielding IC(50) = 1 x 10(-6) M. In contrast, B-II was not a substrate for BoNT/B and exhibited only 25% of the B-I inhibition of BoNT/B. Two additional B-I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 mer; containing B-I amino acids 1-36) and peptide 24 (24 mer; B-I amino acids 16-39). Peptide 24 had a similar inhibitory effect to B-II (ca. 25% of B-I) but peptide 36 was almost 50% as potent as B-I. These findings suggest that the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.
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PMID:Buforin I, a natural peptide, inhibits botulinum neurotoxin B activity in vitro. 1059 94

Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT.
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PMID:Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity. 1130 25

The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).
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PMID:Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization. 1182 15

We have found that hydroxyrthylene (HE) dipeptide analogs of Gln-Arg and Gln-Phe are usually susceptible to acid catalyzed lactonization. The synthesis of substrate-based transition state analog inhibitors of botulinum neurotoxin metalloprotease inhibitors that contain the Gln-Arg or the Gln-Phe HE units is complicated by this facile degradative lactonization.
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PMID:Facile degradative lactonization of Gln-Arg and Gln-Phe hydroxyethylene dipeptide derivatives. 1575 28


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