Iterative l‐Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication

Psilocybe mushrooms produce the psychoactive alkaloid psilocybin from L-tryptophan. A newly identified enzyme, TrpM, mono- and dimethylates L-tryptophan itself, unlike the previously known PsiM enzyme that methylates norbaeocystin. TrpM does not act on tryptamine, indicating a second L-tryptophan-dependent pathway separate from psilocybin biosynthesis. TrpM originated from an ancient duplication of part of the egtDB gene, which codes for an ergothioneine biosynthesis enzyme. This duplicated gene was mostly lost during mushroom evolution but independently re-evolved in various genera. The findings suggest a mechanism where weakly selected genes are preserved by being retained within a widely distributed, conserved metabolic pathway.

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Abstract

Abstract Psilocybe mushrooms are best known for their l ‐tryptophan‐derived psychotropic alkaloid psilocybin. Dimethylation of norbaeocystin, the precursor of psilocybin, by the enzyme PsiM is a critical step during the biosynthesis of psilocybin. However, the “magic” mushroom Psilocybe serbica also mono‐ and dimethylates l ‐tryptophan, which is incompatible with the specificity of PsiM. Here, a second methyltransferase, TrpM, was identified and functionally characterized. Mono‐ and dimethylation activity on l ‐tryptophan was reconstituted in vitro, whereas tryptamine was rejected as a substrate. Therefore, we describe a second l ‐tryptophan‐dependent pathway in Psilocybe that is not part of the biosynthesis of psilocybin. TrpM is unrelated to PsiM but originates from a retained ancient duplication event of a portion of the egtDB gene that encodes an ergothioneine biosynthesis enzyme. During mushroom evolution, this duplicated gene was widely lost but re‐evolved sporadically and independently in various genera. We propose a new secondary metabolism evolvability mechanism, in which weakly selected genes are retained through preservation in a widely distributed, conserved pathway.