Biosynthesis of the Amanita Toxins

Last Updated on Mon, 04/22/2013 - 00:00

The deadly Amanita toxins

Amanita bisporigera (Fig. 1) and its close relatives in genus Amanita, section Phalloideae(such as A. phalloidesA. ocreataA. exitialis, and A. virosa) are responsible for >90% of fatal human mushroom poisonings. These fungi produce amatoxins and phallotoxins. Amatoxins, such as alpha- and beta-amanitin, are specific inhibitors of eukaryotic RNA polymerase II. The structurally related phallotoxins (phalloidin, phallacidin, etc.) bind to actin and stabilize it in the F-actin form.

 

Fig. 1. Amanita bisporigera (Lake Lansing, Michigan)

 

Amatoxins and phallotoxins are bicyclic peptides (Fig. 2). Amatoxins contain eight amino acids and phallotoxins seven. Both contain a Trp-Cys cross bridge (a sulfoxide in amatoxins and a sulfide in phallotoxins), called tryptathionine, that is unique among natural products (May and Perrin, 2007). Both contain multiple hydroxylated amino acids. Phallotoxins contain one D amino acid.

 

Fig. 2. Structures of alpha-amanitin (top), and phallacidin (bottom)

 

Although phallotoxins are fatal when injected, it is the amatoxins that account for the acute toxicity of Amanita mushrooms when eaten. The amatoxins are stable to all forms of cooking and in the digestive tract, and are actively taken up by liver and other cells. Dogs and humans are very sensitive, whereas some animals, such as squirrels, can apparently eat Amanita mushrooms with no ill effects. For more information on the pharmacology of the Amanita toxins, see Benjamin (2005).

We have shown that the Amanita toxins are synthesized on ribosomes, not by nonribosomal peptide synthetases (NRPS) as generally assumed (Hallen et al., 2007; Walton et al., 2004). Although some ascomycete fungi contain more than 15 NRPS genes, A. bisporigera contains none. Amanitin is biosynthesized by the same mechanism in Galerina bisporigera (Luo et al., 2012). POPB, which is clustered with AMA1 in Galerina, is a peptide macrocyclase that converts the 35mer AMA1 propeptide to cyclo(IWGIGCNP) (Luo et al., 2014).

The sequence of Galerina marginata is available at JGI: Galerina genome

 

Fig. 3. Non-fatal species of Amanita. Collected in Mendocino County, California.

 

Natural History of toxin-producing Amanita fungi

Amanita bisporigera is an agaricomycete (mushroom) endemic in North America (Fig. 1). Amanita species, including A. bisporigera, form ectomycorrhizae, which are obligate mutualistic associations with forest trees. The ectomycorrhizal symbiosis is crucial for the health of forest ecosystems worldwide. The ectomycorrhizal fungi Laccaria bicolorand Tuber melanosporum (truffle) have been sequenced (http://genome.jgi-psf.org/Lacbi2/Lacbi2.home.html).

The ectomycorrhizal fungus A. bisporigera is largely deficient in genes encoding proteins that act on the polysaccharides of plant cell walls, such as cellulases, xylanases, pectinases, and other glycosyl hydrolases. The same pattern has been found for the ectomycorrhizal basidiomycete Laccaria bicolor, but saprophytic basidiomycetes such asCoprinopsis cinerea have a full complement of plant cell wall-active enzymes (Nagendran et al., 2009). The lack of cell wall-active enzymes probably reflects two evolutionary imperatives: (1) mycorrhizal fungi obtain their carbon from their plant hosts and therefore do not need these enzymes; (2) cell wall-active enzymes trigger plant defense responses, which would interfere with establishment of the metabolically delicate mycorrhizal symbiosis.

 

Taxonomic distribution of the Amanita toxins

Amatoxins are known to be produced by certain species in three other genera of fungi:GalerinaLepiota, and Conocybe (Benjamin, 1995). These genera are not closely related to each other (Moncalvo et al., 2002). Galerina and Lepiota make amanitins but not phallotoxins (Sgambelleuri et al.,. 2014). Some species of Conocybe also make phallotoxins (Hallen et al., 2003). Many other fungal secondary metabolites also have disjunct phylogenetic distributions (Walton, 2000; Walton et al., 2004). This situation could have arisen by (1) descent from a common ancestor with loss of the toxin genes by intervening taxa; (2) convergent evolution; (3) horizontal gene transfer. We have identified the genes for amanitin production in Galerina marginata, but it is not yet possible to distinguish among these evolutionary scenarios (Luo et al., 2012).

 

Poisonous mushrooms in culture and history

Poisonous mushrooms appear frequently in history, art, movies, and literature (Fig. 4). Tacitus reports that the Roman emperor Claudius was poisoned by his wife, Agrippina, with extracts of poisonous mushrooms obtained from the witch Locusta.

 

Fig. 4. Poisonous mushrooms in literature: The Story of Babar

 

Mushrooom poisoning: clinical aspects

For information on mushroom poisonings, consult your doctor or local poison control center. The North American Mycological Association has more information.

A clinical trial of milk thistle (silibinin, Legalon) to treat amanitin poisoning is taking place at Dominican Hospital in Santa Cruz.

 

Links of interest

The largest collection of fungal genomes is at the Department of Energy Joint Genome Institute.

George Barron's web site on carnivorous and other fungi. Based on his pioneering observations, there are dozens of interesting mushroom natural products waiting to be discovered.

All Things AmanitaThe ultimate resource for Amanita identification and taxonomy, maintained by Drs. Rodham Tulloss and Zhu-Liang Yang.

Mycological Society of America

Tom Volk's fungus web site. Home of the "Fungus of the Month". 

MykoWebA great web site for all things mycological.

Homepage of Heather Hallen-Adams, expert on Amanita taxonomy and discoverer of the Amanita toxin genes, at the University of Nebraska-Lincoln.

The Registry of Mushrooms in Works of Art 

 

Fig. 5. POPB is a peptide macrocyclase

 

References

Benjamin DR (1995) Mushrooms: Poisons and panaceas. New York: WH Freeman and Company. 422 pp.

Hallen HE, R Watling, GC Adams (2003) Taxonomy and toxicity of Conocybe lactea and related species. Mycol Res 107:969-979.

Hallen HE, H Luo, JS Scott-Craig, JD Walton (2007) Gene family encoding the major toxins of lethal Amanitamushrooms. Proc Natl Acad Sci USA 104:19097-19101.

Moncalvo JM, R Vilgalys, SA Redhead, JE Johnson, TY James, MC Aime, V Hofstetter, SJW Verduin, E Larsson, TJ Baroni, RG Thorn, S Jacobsson, H Clemencon, OK Miller, Jr. (2002) One hundred and seventeen clades of euagarics. Mol Phylogenet Evol 23:357–400.

Nagendran S, HE Hallen-Adams, JM Paper, N Aslam, JD Walton (2009) Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei. Fung Genet Biol 46:427-435.

Walton JD (2000) Horizontal gene transfer and the evolution of secondary metabolite gene clusters in fungi: an hypothesis. Fung Genet Biol 30:167–171.

Walton JD, DG Panaccione, HE Hallen (2004) Peptide synthesis without ribosomes. In: Advances in Fungal Biotechnology for Industry, Agriculture and Medicine. J. Tkacz and L. Lange, eds., Kluwer Academic, New York, pp. 127-162.

Wieland T (1986) Peptides of poisonous Amanita mushrooms. Springer, New York

 

Our papers on the Amanita toxins

Hallen HE, H Luo, JS Scott-Craig, JD Walton. (2007) A gene family encoding the major toxins of lethal Amanita mushrooms. Proc Natl Acad Sci 104:19097-19101.

Luo H, HE Hallen-Adams, JD Walton (2009) Processing of the phalloidin
proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes. J Biol Chem 284:18070-18077.

Luo H, HE Hallen-Adams, JS Scott-Craig, JD Walton (2010) Co-localization of amanitin and a candidate toxin-processing prolyl oligopeptidase in Amanita basidiocarps. Eukaryotic Cell 9: 1891–1900.

Walton JD, HE Hallen-Adams, H Luo (2010) Ribosomal biosynthesis of the cyclic peptide toxins of Amanitamushrooms. Biopolymers (Peptide Science) 94:659-664.  

Luo H, HE Hallen-Adams, JS Scott-Craig, JD Walton (2012) Ribosomal biosynthesis of alpha-amanitin in Galerina marginata. Fung. Genet. Biol. 49:123-129.

Arnison, P. G., M. J. Bibb, G. Bierbaum, A. A. Bowers, G. Bulaj, J. A. Camarero, D. J. Campopiano, J. Clardy, P.D. Cotter, D.J. Craik, E. Dittmann, S. Donadio, P. C. Dorrestein, K.-D. Entian, M. A. Fischbach, J.S. Garavelli, U. Göransson, C.W. Gruber, D.H. Haft, T. K. Hemscheidt, C. Hertweck, C. Hill, A.R. Horswill, M. Jaspars, W.L. Kelly, J. P. Klinman, O. P. Kuipers, A. J. Link, W. Liu, M. A. Marahiel, D. A. Mitchell, G. N. Moll, B. S. Moore, S. K. Nair, I. F. Nes, G. E. Norris, B. M. Olivera, H. Onaka, M. L. Patchett, M. J.T. Reaney, S. Rebuffat, R. Paul Ross, H.-G. Sahl, E. W. Schmidt, M. E. Selsted, K. Severinov, B. Shen, K. Sivonen, L. Smith, T. Stein, R. E. Süssmuth, J. R. Tagg, G.-L. Tang, J. C. Vederas, C. T. Walsh, J. D. Walton, J. M. Willey,  and W. A. van der Donk (2013) Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30:108-160. PMID: 23165928

Riley, R., A. Salamova, D. Brown, L.G. Nagy, D. Floudas, B. Held, A. Levasseur, V. Lombard, E. Morin, R. Otillar, E. Lindquist, H. Sun, K. LaButt, J. Schmutz, D. Jabbour, H. Luo, S. Baker, A. Pisabarro, J.D. Walton, R. Blanchette, B. Henrissat, F. Martin, D. Cullen, D. Hibbett, and I.V. Grigoriev (2014). Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white rot/brown rot paradigm for wood decay fungi. Proc. Natl. Acad. Sci. USA 111:9923-9928. PMID: 24958869.

Sgambelluri, R.M., S. Epis, D. Sassera, H. Luo, E.R. Angelos, and J.D. Walton (2014) Profiling of amatoxins and phallotoxins in the genus Lepiota by liquid chromatography combined with UV absorbance and mass spectrometry. Toxins 6:2336-2347. PMID: 25098279

Luo, H., S.Y. Hong, R.M. Sgambelluri, E. Angelos, X. Li, and J.D. Walton (2014) Peptide macrocyclization catalyzed by a prolyl oligopeptidase involved in α-amanitin biosynthesis. Chem Biol. 21:1610-1617.

 

Documents

Hallen PNAS Amanita genes.pdf

Luo et al. Co-localization of amanitin and POP in basidiocarps

Luo et al. Ribosomal biosynthesis of amanitin in Galerina

Luo et al. 15N amanatin in Galerina