Evaluation of TrpM and PsiD substrate promiscuity reveals new biocatalytic capabilities

N-methylated tryptamines like psilocybin and DMT show promise as treatments for mental health disorders, driving interest in biosynthetic production. This work characterized two enzymes from tryptamine biosynthesis: TrpM, a tryptophan N-methyltransferase from Psilocybe serbica, and PsiD, a decarboxylase from the psilocybin pathway. TrpM was able to N-methylate 4-hydroxytryptophan, a non-native amino acid. However, incorporating TrpM into a functional psilocybin pathway was blocked because PsiD could not use N,N-dimethyl-4-hydroxytryptophan as a substrate under the tested conditions, despite acting on N-methylated and 4-hydroxylated tryptophan derivatives separately. These findings expand the known substrates for TrpM and PsiD, increasing the diversity of tryptamine biosynthetic products.

Study at a glance

Abstract

Abstract N ‐methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N ‐dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for mental health disorders. The clinical relevance of these compounds has driven scientists to develop biosynthetic production routes to a number of tryptamine drug candidates, and efforts are ongoing to expand and further develop these biosynthetic capabilities. To that end, we have further characterized the substrate preferences of two enzymes involved in tryptamine biosynthesis: TrpM, a tryptophan N ‐methyltransferase from Psilocybe serbica , and PsiD, the gateway decarboxylase of the psilocybin biosynthesis pathway. Here, we show that TrpM can N ‐methylate the non‐native amino acid substrate, 4‐hydroxytryptophan, a key intermediate in the Escherichia coli ‐based recombinant psilocybin biosynthesis pathway. However, the ability to incorporate TrpM into a functional psilocybin biosynthesis pathway was thwarted by PsiD's inability to use N,N ‐dimethyl‐4‐hydroxytryptophan as substrate, under the culturing conditions tested, despite demonstrating activity on N ‐methylated and 4‐hydroxylated tryptophan derivatives individually. Taken together, this work expands upon the known substrates for TrpM and PsiD, further increasing the diversity of tryptamine biosynthetic products.