The Second Methylation in Psilocybin Biosynthesis Is Enabled by a Hydrogen Bonding Network Extending into the Secondary Sphere Surrounding the Methyltransferase Active Site
Jesse Hudspeth, Kai Rogge, Tobias Wagner, Maximilian Müll, Dirk Hoffmeister, Bernhard Rupp, Sebastiaan Werten
First published: 16 October 2024 https://doi.org/10.1002/cbic.202400497
Graphical Abstract
M at work: PsiM is the methyltransferase that completes psilocybin biosynthesis in magic mushrooms. Crystal structures and kinetic analyses of the wild type enzyme and two engineered variants provided profound insight into why the residue Asn247 is key for PsiM’s capability to dimethylate.

The Psilocybe cubensis SAM-dependent methyltransferase, PsiM, catalyzes the last step in the biosynthesis of psilocybin. Likely evolved from monomethylating RNA methyltransferases, PsiM acquired a key amino acid exchange in the secondary sphere of the active site, M247 N, which is responsible for its capacity to dimethylate. Two variants, PsiMN247M and PsiMN247A, were generated to further examine the role of Asn247 for mono- and dimethylation in PsiM. Our structural and kinetic analyses of the variants suggest that Asn247 is essential to allow enough space in the active site for multiple methylations while also participating in a network of hydrogen bonds that stabilizes secondary structure elements in the immediate vicinity of the active site for optimal methylation of norbaeocystin.