Iterative l‐Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication

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.

Enzymatic Route toward 6‐Methylated Baeocystin and Psilocybin

“…A similar enzyme, Ybs of Aspergillus nidulans , preferentially trimethylates l ‐tyrosine, but also l ‐histidine, l ‐DOPA, and l ‐phenylalanine . The phylogenetically related Psilocybe serbica methyltransferase TrpM, which is not part of 1 biosynthesis, dimethylates the amino groups of l ‐tryptophan, l ‐tyrosine, and l ‐phenylalanine, but transfers a third methyl group only very poorly . Despite their being substrate flexible, kinetics and affinity determine the number of methyl groups transferred by the above enzymes, whereas the number of catalytic cycles was independent of the acceptor compound.…”

“…For heterologous production of S. cerevisiae TRP5, E. coli KRX transformed with pJF56 was used. Production of E. coli S ‐adenosylhomocysteine nucleosidase and adenine deaminase followed a described procedure . The above enzymes were purified by immobilized metal‐affinity chromatography on a His Trap FF Crude column (1 mL bed volume), fitted to an Äkta Pure 25 FPLC instrument (GE Healthcare).…”

Taking Different Roads: l‐Tryptophan as the Origin of Psilocybe Natural Products

“…Surprisingly, chromatographic analysis identified another set of l ‐tryptophan‐derived natural products in this species, l ‐abrine and N , N ‐dimethyl‐ l ‐tryptophan (Scheme 1). [47] This somewhat surprising finding raised the question: Which enzyme would catalyze this L‐tryptophan modification and how does it relate to psilocybin biosynthesis?…”

“…Instead, TrpM shows a close relationship to the above‐mentioned EgtD. The trpM gene originates from a very ancient partial duplication of the egtDB gene [47] . In the course of basidiomycete evolution, most fungi lost trpM , while P. serbica retained the gene.…”

“…TrpM's substrate specificity supports this biosynthetic feature: it is strictly specific for l-tryptophan and did not accept tryptamine or 4-hydroxytryptamine, the very intermediates of psilocybin biosynthesis that could be potentially converted to psilocin. [47] Hence, TrpM works completely separately from the psilocybin pathway, and the two routes mutually avoid interfering with each other (Scheme 1).…”