9FMJ

PsiM N247M in complex with sinefungin and baeocystin

Summary for 9FMJ

• Entry DOI: 10.2210/pdb9fmj/pdb
• Descriptor: Psilocybin synthase, SINEFUNGIN, Baeocystin, ... (5 entities in total)
• Functional Keywords: methyltransferase, rossmann fold, complex, biosynthetic protein
• Biological source: Psilocybe cubensis
• Total number of polymer chains: 1
• Total formula weight: 36629.19

Authors

Hudspeth, J.,Rupp, B.,Werten, S. (deposition date: 2024-06-06, release date: 2024-10-23, Last modification date: 2024-12-11)

Primary citation

Hudspeth, J.,Rogge, K.,Wagner, T.,Mull, M.,Hoffmeister, D.,Rupp, B.,Werten, S.
The Second Methylation in Psilocybin Biosynthesis Is Enabled by a Hydrogen Bonding Network Extending into the Secondary Sphere Surrounding the Methyltransferase Active Site.
Chembiochem, 25:e202400497-e202400497, 2024

PubMed 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 and PsiM, 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.

PubMed: 39413044
DOI: 10.1002/cbic.202400497

Experimental method

X-RAY DIFFRACTION (0.95 Å)