A Revised Model for Muscarine Biosynthesis Involving Lysine Trimethylation.

l-(+)-Muscarine is a widespread fatal toxin produced by various mushrooms that pose a severe threat to human health when they are mistaken for edible species. Apart from a single 1970s study that assumed l-glutamate and pyruvate were the building blocks of this unusual quaternary amine, surprisingly little is known about the toxin's biogenesis. We used Collybia rivulosa (syn. Clitocybe rivulosa), a mushroom notorious for producing muscarine, as our model for stable isotope incorporation experiments and subsequent extensive mass spectrometric analysis. Our results provide unambiguous evidence that the backbone of muscarine is assembled from two amino acids, l-lysine and l-alanine. Furthermore, we found that iterative ε-methylation of non-protein-bound l-lysine is the biosynthetic gateway step that yields ε-N,N,N-trimethyl-l-lysine. This methylation is specific to fungi that produce muscarine. Despite a substrate overlap with the biosynthesis of l-carnitine, we demonstrate that these two pathways are distinct. Our results provide compelling insight into the biogenetic origin of muscarine and fundamentally revise the previous biosynthetic model for this infamous toxin. The revised biosynthesis model lays the foundation to discover as yet unknown muscarine-like metabolites that are potentially toxic as well or pharmacologically relevant.