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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

ChemBioChem October 16, 2024 DOI: 10.1002/cbic.202400497 (opens in new tab)

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Study at a glance

AI-extracted from the abstract
Characteristics Experimental study Peer reviewed
Population PsiM enzyme variants from Psilocybe cubensis
Topics Psilocybin
Keywords Enzyme Methyltransferase Methylation Active site Stereochemistry Ternary complex Biosynthesis Substrate aquarium Cofactor Biochemistry
Citations 1
Key findings The residue Asn247 in PsiM is essential for both creating space for dimethylation and stabilizing the active site structure for optimal methylation of norbaeocystin.

Abstract

Abstract 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, PsiM N247M and PsiM N247A , were generated to further examine the role of Asn247 for mono‐ and dimethylation in PsiM. Herein, we present the kinetic profiles of both variants and crystal structures at resolutions between 0.9 and 1.0 Å. Each variant was crystallized as a ternary complex with the non‐methylated acceptor substrate, norbaeocystin and S ‐adenosyl‐ l ‐homocysteine, and in a second complex with the cofactor analog, sinefungin, and the monomethylated substrate, baeocystin. Consistent with the inability of the variants to catalyze a second methyl transfer, these structures reveal catalytically non‐productive conformations and a high level of disorder of the methylamine group of baeocystin. Additionally, both variants exhibit destabilization in the β5‐β7 sheets and a conserved β‐turn of the core Rossmann fold, causing 20‐fold reduced substrate binding and 2‐fold lower catalytic efficiency even with norbaeocystin. 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.

Comparable studies

Other experimental studies on psilocybin, most cited first.

Study Year Design Participants
Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action Healthy human volunteers 1998 Experimental study
Validation of the revised Mystical Experience Questionnaire in experimental sessions with psilocybin Participants in controlled laboratory experiments receiving a moderate to high oral... 2015 Validation study using pooled data from five laboratory experiments n = 184
Broadband Cortical Desynchronization Underlies the Human Psychedelic State Healthy human participants 2013 Experimental study
Long-term follow-up of psilocybin-assisted psychotherapy for psychiatric and existential distress in patients with life-threatening cancer Participants with cancer-related psychiatric distress who completed a parent randomized... 2020 Long-term within-subjects follow-up analysis n = 15
Enzymatic Synthesis of Psilocybin 2017 Laboratory study

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