The Second Methylation in Psilocybin Biosynthesis Is Enabled by a Hydrogen Bonding Network Extending into the Secondary Sphere Surrounding the Methyltransferase Active Site
ChemBioChem – October 16, 2024
Source: OpenAlex
Summary
A single amino acid change in the *Psilocybe cubensis* enzyme PsiM, a methyltransferase, enables the crucial dimethylation step in psilocybin biosynthesis. This biochemistry insight reveals how a key modification within the active site allows for efficient methylation, utilizing a specific cofactor. Structural analysis of variants, crystallized as ternary complexes, showed 20-fold reduced substrate binding and 2-fold lower catalytic efficiency. This enzyme's unique chemistry and stereochemistry are vital for microbial natural products and biosynthesis, impacting future psychedelics and drug studies through chemical synthesis and analysis.
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.