The Peptides of Amanita Mushrooms

The cyclic 11-peptide contains mainly normal amino acids, one D-alanine and one L-a-aminobutyric acid, but, strikingly, not less than seven iV-methyl groups and a quite uncommon amino acid, a C9-compound (4R)-4[(F)-2-butenyl]-4-methyl-N-methyl-L-threonine. 

The total synthesis of cyclosporin A has been described by R.M. Wenger [38] biosynthetically it is produced by adding appropriate amino acids to the fermentation with the fungus [39] or to cell free preparations therefrom [40]. Various cyclosporins with different activities have been obtained. The activation and connection of the amino acids occurs, as recognized in the synthesis of gramicidin S (p. 208), by the thiotemplate mechanism the methyl groups are introduced into the already formed peptide bonds by S-adenosyl-methionine. 

The investigation of the deadly poison of Amanita mushrooms was initiated at the beginning of this century in the USA. At Johns Hopkins University in Baltimore, MD, the bacteriologist William W. Ford attempted the isolation of the toxin, amanitatoxin , and succeeded in obtaining a preparation containing about 5% of the fatal agent without recognizing its peptide nature. The efforts were resumed in the thirties in the laboratory of Heinrich Wieland in Munich, 

Bavaria, where in 1937 Ulrich Wieland and Feodor Lynen were able to crystallize for the first time a substance which was called phalloidin that rapidly killed mice after intraperitoneal application. Three years later Heinrich Wieland and Rudolf Hallermayer crystallized from Amanita phalloides extracts a second toxin, amanitin , which with smaller doses killed the experimental animals only after several days Uke a deadly dish of A, phalloides kills humans. Bernhard Witkop (Plate 49) 1940 in H. Wieland s laboratory recognized that phalloidin is a peptide and isolated a new imino acid, allo-hydroxyproline. Then the Second World War interrupted Amanita research which was resumed in Heidelberg by one of the present authors only ten years later and continued in Mainz, Frankfurt and again in Heidelberg through the following decades. 

---

The history of the major achievements concerning the isolation, structure elucidation, chemistry, and biological activity of the cyclic peptide principles of A. phalloides was comprehensively covered mainly by T. Wieland, a man of great merit particularly in this branch of natural products research. He dealt with the subject in several review articles 10,87,94,98-101) and more recently in an excellent book on the peptides of poisonous Amanita mushrooms 86). The number of references cited in the book exceeded 750, which gives a rough idea of the proportion of research carried out by chemists on this topic. The scope of the present chapter enables us only to summarize briefly the final results of these interesting studies. 

Mushrooms of the Hymenomycetes provide a good source of secondary metabolites having the indole moiety incorporated into their molecular structures, as in the case of Amanita peptide alkaloids. Several of the indole alkaloids reveal strong physiological activity and have been found to be an important pharmaceutical material. 

Phalloidin and phallacidin are cyclic peptides from the mushroom Amanita phalloides that stabilize F-actin. Phalloidin binds to residues 114-118 of an actin protomere and blocks nucleotide exchange without interfering with nucleotide hydrolysis. It enhances the rate of nucleation as well as that of elongation. It slowly penetrates the cell membrane and is used for immunocytochemical localization of F-actin. 

One peptide toxin from the mushroom Amanita phalhides, a-amanitin, is a specific differential inhibitor of the eukaryotic nuclear DNA-dependent RNA polymerases and as such has proved to be a powerful research tool (Table 37-2). a-Amanitin blocks the translocation of RNA polymerase during transcription. 

Other species of amanita are among the deadliest fungi known. Polypeptide-like toxins in Amanita phalloides, or death cup, can prove fatal or at the very least can cause permanent liver and kidney damage. These mushrooms are common in the temperate climates of Europe and North America. They are responsible for the majority of what is called "slow" mushroom poisoning in the U.S. In fact, it was only a few years ago that the local newspaper reported a case of amanita poisoning within SLO county. Mushroom poisoning is known as mycetism. In addition the amanita also contain bufotenine which has CNS effects. See the ASIDE which discusses the peptide poisons. 

It also should be noted that within the genus Amanita there exist several species of deadly mushrooms. They are among the small number of mushroom species the ingestion of which can prove fatal. These species include Amanita phalloides and Amanita viA, both of which contain small peptides called amanitins that inactivate RNA polymerase and cause irreversible damage to liver function. 

O. 5 mg kg causes fiill protection of mice from death by phalloidin phallotoxins) after injection about 1 h before or, at the latest, simultaneously with 5 mg kg of the toxin. Antamanide is a competitive inhibitor of the transport system for phallotoxins and amatoxins in the parenchyma cells of the liver [Ih. Wieland, Peptides of Poisonous Amanita Mushrooms, Springer Series in Molecular Biology, Springer Ver-lag, Berlin, New York, 1986 K. Munster etal., Biochem. Biophys. Acta 1986, 860,91]. 

The extremely poisonous green mushroom Amanita phalloides contains a number of cyclic peptides classified as phallotoxins (heptapeptides), ama-toxins (octapeptides), and a decapeptide antagonist called antamanide (Wieland and Wieland, 1972). Crystal structure analyses have been completed on jS-amanitin, on antamanide, and on a biologically active analog, both com-plexed and uncomplexed. 

Amatoxins, besides the already mentioned Amanita species, were found also in other mushrooms noted for their toxicity the inconspicuous Galerina species, G. marginata and G. venenata and the Lepiota mushrooms as well, contain these peptides, synthesized not in the carpophore but in the mycelia, most probably through the non-ribosomal pathway. Several hundred more mushrooms have been examined, for instance with the help of the newsprint test, and it is unlikely that further amatoxin accumulating species will be found. Phallotoxins have been detected only in Amanita toadstools. 

A cyclic peptide that neutralizes the effect of phalline B, a toxin (also a cyclic peptide) present in the deadly mushroom Amanita phalloides. 

Poisoning and sometimes death from eating (unidentified) mushrooms is well known. In particular, Amanita sp. are particularly dangerous, with much emphasis on the death cap fungus , Amanita phalloides.24 The best known toxins are the amatoxins and phallotoxins, which are complex, bicyclic peptides. An unusual feature relates to sulfur a tryptophan (or substituted tryptophan) unit is linked to a cysteine sulfur at the carbon atom next to the NH group of the pyrrole ring, forming the unit, -CH2-S-C(NH)=C, e.g. in... 

The poisonous components of the most deadly mushroom Amanita phalloides (the Death Cap) are bicyclic heptapeptides which have an additional covalent bond that connects the ( -sulfur atom of an l-cysteine residue with the carbon atom in position 2 of the indole ring of the L-tryptophan. Phalloidin (or phalloidine) is the most abundant member of a whole family of related cyclic heptapeptides called phallotoxins (for a review, see Wieland1 1). These poisonous peptides, therefore, contain a cross-linking moiety consisting of L-tryptophan coupled to L-cysteine, designated tryptathionine (1), alternatively called 5-(trypto-phan-2-yl)cysteine or 2-(L-3-alanylsulfenyl)-L-tryptophan (Scheme 1). 

Amanita family of mushrooms which contains several toxic peptides helonging to the amatnxin and phalloioxin groups. 

Substances related to MSG and purine 5 -ribonucleotides include peptides, amino acids (e.g. cysteine, homocysteine, cysteine S-sulfonic acid, aspartic acid, a-amino adipic acid, a-methyl glutamic acid, tricholomic acid, ibotenic acid), pyrrolidone carboxylic acid, 3-methyl thiopropyl amine, and others [2, 10], They are of less commercial interest than MSG, IMP, and GMP. Chemical structures of some of these substances are depicted in Fig. 3.53. Relative umami effects of some are shown in Tab. 3.49. Tricholomic acid and ibotenic acid have been found in the mushrooms Tricholoma muscarium and Amanita stroboliformis, respectively. 

The green death cap Amanita phalloides produces two families of toxic peptides, the amatoxins and the phallotoxins. Amatoxins are by far the more dangerous toxins and account for all fatalities occurring in human mushroom poisoning. 

---