peptide toxins are found in venom from animal species, we will focus on the larger-protein toxins produced by bacteria and some plants. For the purpose of this appendix, we define a toxin as any protein that has a deleterious effect on health and fitness when ingested.

Seven immunologically distinct and extremely potent protein neurotoxins are produced by C. botulinum: A, B, C, D, E, F, and G. Types A, B, and E are most frequently associated with human disease, and types F and G are less often reported. Types C and D are associated with disease in fowl. The BoNTs are all expressed as single polypeptides that are posttranslationally proteolyzed to give a heavy chain and a light chain that are linked by a disulfide bond. The heavy chain is responsible fo r toxin adherence to the cell surface and translocation of the light chain into the cytosol. The light chain contains the zinc protease active site that is responsible for cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE). BoNTs bind ganglioside receptors on the neuronal cell surface in the presynaptic terminals and, by their action as metalloendoproteases, selectively cleave proteins involved in the neuroexocytosis apparatus; this results in inhibition of acetylcholine release at the myoneural junctions and later flaccid paralysis that can lead to respiratory arrest.

The enzymatic active site of these toxins has been well characterized. A crystal structure of BoNT/A demonstrated that zinc is coordinated by the HisGluXXHis motif in the active site alpha helix through His-222 and His-226 and by Glu-261 (Lacy, Tepp et al. 1998). Site-directed mutagenesis experiments demonstrated the importance of these residues for toxin activity (Fujii, Kimura et al. 1992). Because the zinc binding site of this class of toxins is so well conserved (Fujii, Kimura et al. 1992) it would seem possible to predict from the gene sequence whether a bacterium is able to produce active toxin. However, data that argue against the use of BoNT sequence to predict activity comes from Agarwal et al. (Agarwal, Binz et al. 2005), who determined the structure of BoNT/E light chain with single amino acid substitutions outside of the canonical active site in regions adjacent to the catalytic residues. They found that the relatively minor change of glutamate to glutamine at position 335 rendered the enzyme unable to bind zinc. Other mutations in these regions also had large effects on the activity of the toxin. These findings serve to emphasize that biological toxin activity not only depends on the primary sequence but also on the three-dimensional structure of the folded protein.



Bacillus anthracis is a gram-positive, spore-forming rod that is an etiological agent of pulmonary, cutaneous, and gastrointestinal anthrax. In cases of pulmo-

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