Atomic-resolution (0.9 Å) crystal structures of PsiM at multiple reaction stages reveal the SAM-dependent dimethylation mechanism and show that its substrates physicochemically mimic RNA, while structural and phylogenetic analyses indicate PsiM derives from METTL16-family m6A writers. The study also shows inherent limitations of the ancestral monomethyltransferase scaffold that reduce psilocybin assembly efficiency and prevent trimethylation to aeruginascin, informing bioengineering efforts to create improved psilocybin variants.
Psilocybin, the natural hallucinogen produced by Psilocybe (“magic”) mushrooms, holds great promise for the treatment of depression and several other mental health conditions. The final step in the psilocybin biosynthetic pathway, dimethylation of the tryptophan-derived intermediate norbaeocystin, is catalysed by PsiM. Here we present atomic resolution (0.9 Å) crystal structures of PsiM trapped at various stages of its reaction cycle, providing detailed insight into the SAM-dependent methylation mechanism. Structural and phylogenetic analyses suggest that PsiM derives from epitranscriptomic N6-methyladenosine writers of the METTL16 family, which is further supported by the observation that bound substrates physicochemically mimic RNA. Inherent limitations of the ancestral monomethyltransferase scaffold hamper the efficiency of psilocybin assembly and leave PsiM incapable of catalysing trimethylation to aeruginascin. The results of our study will support bioengineering efforts aiming to create novel variants of psilocybin with improved therapeutic properties.
Hudspeth and colleagues introduce PsiM as the SAM-dependent methyltransferase that performs the final biosynthetic step generating psilocybin from norbaeocystin via the monomethylated intermediate baeocystin. The authors situate psilocybin in a therapeutic context, noting its clinical promise and recent efforts to produce it biotechnologically, and highlight that the four genes required for the pathway (psiD, psiH, psiK and psiM) have enabled heterologous production in several microbial hosts. This study set out to determine high-resolution crystal structures of PsiM at successive stages of its catalytic cycle, to characterise its enzymology and substrate specificity, and to investigate its evolutionary origins. The aims included explaining why PsiM dimethylates its small-molecule substrate, whether it can perform a third methylation to yield aeruginascin, and how the enzyme’s structure relates to related RNA methyltransferases, with the practical goal of informing bioengineering of psilocybin analogues.
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The investigators expressed recombinant PsiM from Psilocybe cubensis in Escherichia coli and produced several preparations for structural and biochemical work. For crystallography, full-length PsiM (with and without removable affinity tags) was purified by nickel affinity and size-exclusion chromatography, tag-cleaved where indicated, complexed with coenzyme analogues and substrate or products, flash-frozen, and stored prior to crystallisation. A variety of complexes were prepared by adding twofold molar excesses of SAH or sinefungin and fivefold excesses of norbaeocystin or baeocystin. Crystallisation employed hanging- and sitting-drop vapour diffusion at 4 °C and produced multiple crystal forms; orthorhombic, monoclinic and tetragonal forms were obtained for different complexes. X-ray diffraction data were collected at ESRF beamlines at 100 K and processed with standard pipelines (xia2/DIALS or XDS). Structures were solved by molecular replacement and refined using Coot and Phenix, with several complexes solved at atomic resolution (<= 0.94 Å). Enzymatic assays used purified PsiM (and an engineered N247M mutant) in 50 mM Tris buffer (pH 8.4) with SAM and acceptor substrates (norbaeocystin, baeocystin or psilocybin). Assays for activity, temperature and pH optima, and kinetics were carried out with UHPLC-MS detection; kinetics employed 500 nM PsiM, 1 mM SAM and varied acceptor concentrations (100 µM–1 mM) with timepoints up to 600 s. Isothermal titration calorimetry (ITC) at 25 °C measured binding of SAH, sinefungin and substrates to PsiM under a single-site binding model. Finally, sequence searches, multiple alignment (MAFFT) and maximum-likelihood phylogenetic analysis (MEGA11, JTT model) were used to place PsiM among METTL16 and RlmF family methyltransferases.
Atomic-resolution crystal structures of PsiM were obtained for multiple ligand states. Two crystal forms of the PsiM–SAH–norbaeocystin ternary complex were solved at 0.91 Å (orthorhombic, single molecule per ASU) and 1.18 Å (monoclinic, two molecules per ASU). Additional high-resolution structures include PsiM–sinefungin–norbaeocystin (0.89 Å), PsiM–SAH–baeocystin (0.93 Å), PsiM–sinefungin–baeocystin (0.92 Å) and PsiM–SAH–psilocybin (0.94 Å). The fold is a canonical class I Rossmann methyltransferase with an additional N‑terminal jaw-like domain and a substrate recognition loop (SRL) that closes over the bound ligand. The active site geometry explains key catalytic features. Norbaeocystin’s amino group is held by hydrogen bonds to the backbone carbonyl of Pro184 (N–O 2.8 Å) and the side-chain oxygen of Asn183 (N–O 2.9 Å), positioning the nitrogen lone pair towards the transferable methyl of SAM. Mutation N183A abolishes catalysis, consistent with its central role. Substrate recognition is highly specific: the indole is sandwiched between Phe202 and Met217, and the phosphate is gripped by Arg75 and Arg281. The SRL (residues ~189–221) forms a lid that sequesters the substrate; in the absence of substrate portions of the SRL are disordered and the active site opens. ITC shows sequential binding: SAH binds PsiM alone (Kd 66 ± 49 µM), whereas binding of norbaeocystin (Kd 35.7 ± 5.4 µM) or baeocystin (Kd 77.2 ± 3.5 µM) requires coenzyme presence, supporting an ordered mechanism where coenzyme binds first and substrate second. Crystal structures reveal two principal ligand binding modes: an engaged mode with the ethylamide nitrogen at the catalytic centre, and a disengaged mode with the indole ring shifted away, which likely facilitates deprotonation and product release. In the orthorhombic PsiM–SAH–norbaeocystin complex the main binding mode has 64% occupancy while an alternative conformation accounts for 36% occupancy. Kinetic measurements indicate the first methyl transfer is faster than the second: kcat values are 0.11 ± 0.01 min^-1 (first transfer) and 0.06 ± 0.01 min^-1 (second transfer). Michaelis constants are 575 ± 100 µM for norbaeocystin and 492 ± 154 µM for baeocystin, yielding catalytic efficiencies of 0.191 and 0.122 nM min^-1, respectively. Chromatography shows accumulation of the monomethylated intermediate baeocystin in vitro, consistent with lower affinity for baeocystin in some ternary complexes (for example, substrate occupancy falls to 61% in the PsiM–sinefungin–baeocystin structure). Structural and phylogenetic analyses link PsiM to the METTL16 family of m6A RNA methyltransferases. Dali searches returned METTL16 catalytic-domain structures with high similarity (Dali Z-scores 25–35); the best match (PDB 6DU4) superposes with a Cα RMSD of 1.5 Å over 260 residues. Sequence-based phylogeny places PsiM within the METTL16 family, which branches from ribosomal RlmF m6A methyltransferases. Superposition of substrate pockets shows that norbaeocystin’s phosphate occupies a position analogous to the RNA phosphate and the indole ring mimics adenine, supporting substrate mimicry as an evolutionary driver. Mechanistic and mutational findings identify a single-residue adaptation that enabled dimethylation. In PsiM Asn247 sits behind Asn183 and can hydrogen-bond to a rotated Asn183, creating space for the first methyl group; in METTL16 orthologs the equivalent position is occupied by bulky hydrophobic residues (Leu/Met) that would hinder Asn movement. The reverse mutation N247M in PsiM preserves monomethylation but abolishes efficient psilocybin (dimethyl) formation, consistent with the hypothesis that (M/L)→N substitution allowed dimethyltransferase activity to evolve. Finally, biochemical assays do not support PsiM-mediated trimethylation to aeruginascin. Under optimised conditions (temperature window 20–30 °C, pH 8.4), PsiM converted norbaeocystin or baeocystin to psilocybin but did not produce detectable aeruginascin in vitro. The authors attribute this inability to the rigid NPPF motif and constrained backbone that prevent the conformational rearrangements needed to accommodate a dimethylated substrate for a third methyl transfer.
The researchers interpret their atomic-resolution structures as supporting a proximity-and-desolvation mechanism in which the active site enforces close approach of nucleophile and methyl donor while excluding solvent. Modelled positions of the transferable methyl group place it 1.7–1.8 Å from the nucleophilic nitrogen with favourable S–C–N angles, consistent with stabilisation of the transition state. Several CH hydrogen bonds between coenzyme and protein are noted; chalcogen bonding to the coenzyme sulphur was not observed. Alternative ligand binding modes revealed by the structures are proposed to have functional roles: the fully engaged mode aligns the nucleophilic nitrogen for methyl transfer, whereas the disengaged mode likely facilitates initial substrate capture, amine deprotonation prior to the transition state, and subsequent product release. The SRL, which appears to have evolved from an RNA-recognition motif, acts as a specialised lid that sequesters the small-molecule substrate while the Rossmann core and catalytic centre are conserved. On evolution, the authors argue that PsiM arose from METTL16/RlmF-like m6A RNA monomethyltransferases through substrate mimicry: the phosphorylated tryptamine substrate mimics RNA geometry and phosphates, allowing an ancestral RNA methyltransferase scaffold to be repurposed for small-molecule dimethylation. A single amino-acid change—introduction of Asn at the position equivalent to 247—relieved steric restriction on the catalytic Asn and permitted a second methylation. Despite this innovation, PsiM remains an inefficient dimethyltransferase: the second methyl transfer is slower, and the enzyme must release the monomethylated intermediate to exchange SAH for SAM, creating a kinetic bottleneck that the authors describe as a likely local evolutionary optimum. Implications discussed include the use of PsiM structures as templates for understanding METTL16 catalysis and for designing METTL16 inhibitors, given the substrate-mimicry parallels. The authors also highlight bioengineering applications: the structures indicate which indole substitutions (positions 6 and 7) might be accommodated by wild-type PsiM and caution that the phosphate moiety is critical for binding. Differences between PsiM and METTL16 are acknowledged, notably lack of conservation of regulatory K-loop residues and differing ligand-binding order, and the authors note that SAM-containing ternary complexes were not obtained because SAM would allow the reaction to proceed, limiting direct observation of the transition state. Overall, the findings are presented as providing mechanistic, evolutionary and practical insight useful for enzyme engineering and for informing future studies of related epitranscriptomic methyltransferases.
In the present study, we describe the reaction mechanism behind the biosynthesis of psilocybin and present the highest-resolution crystal structures for any methyltransferase to date. The structures are consistent with the "proximity and desolvation" model, which states that the active site of a methyltransferase forces the reactive groups into close proximity while excluding any interfering solvent molecules from their environment. Formation of so-called near-attack Fig.| Consecutive stages of the reaction cycle. The NPPF motif of PsiM is shown as a stick model (beige). SAH (yellow), sinefungin (light/dark green) and (nor) baeocystin/psilocybin (pink/purple) are represented as ball-and-stick models, with the darker colour representing the main conformation. Corresponding OMIT maps are shown in grey (2mF o -DF c with both ligands omitted from the model, maps contoured at 1.0 σ). Key hydrogen bonds are shown as dashed lines. Each model is rotated slightly differently so as to clearly reveal its relevant properties. a The PsiM-SAH-norbaeocystin complex (PDB 8PB4, 0.91 Å), revealing an alternative substrate conformation potentially involved in amine deprotonation. b The PsiM-sinefunginnorbaeocystin complex (PDB 8PB5, 0.89 Å), a model for the state of the complex prior to the first methyl transfer. c The PsiM-SAH-baeocystin complex (PDB 8PB6, 0.93 Å), revealing the state of the active site immediately after the first methyl transfer. d The PsiM-sinefungin-baeocystin complex (PDB 8PB7, 0.92 Å), approximating the situation immediately before the second methyl transfer. e The PsiM-SAH-psilocybin complex (PDB 8QXQ, 0.94 Å). f Superposition of the structures shown in panels a-e. Interactions shown as dashed lines belong to the main conformation of the PsiM-SAH-norbaeocystin complex (a). Two distinct ligand positions relative to the binding site have been emphasised by means of the colours magenta and cyan. conformers (NACs), electronic pre-organisation, cratic effectsand compactionhave often been described as likely contributing factors, whereas more recent studies highlight the role of pre-transition state tetrel bond formation, chalcogen bondingand CH hydrogen bonding 52 in the process. Although the (pre-)transition state cannot be directly observed in structural studiesthe ternary complexes under investigation do not contain SAM as it would allow the reaction to proceedour atomic-resolution data prompt a detailed analysis of the active site geometry in the presence of two closely related coenzyme analogues. Modelling SAM on the basis of the experimentally determined coordinates of SAH or sinefungin places the transferable methyl group at 1.7-1.8 Å from the fully engaged nucleophilic nitrogen, i.e. equidistant from the nitrogen and the sulfur, with S-C-N angles of 167°and 173°, respectively (Fig.). Thus, the ligand binding mode observed in our structures would be expected to stabilise the transition state and favour shortening of the initial tetrel bond between the nucleophile and the methyl carbon. Consistent with observations made for other methyltransferases, several CHhydrogen bonds link the coenzyme to PsiM (Fig.). On the other hand, we do not observe any chalcogen bondinginteractions between PsiM and the sulfur atom of the coenzyme. Intriguingly, the atomic-resolution structures reveal a number of alternative substrate and product binding modes. Roughly, two types of ligand positioning with respect to the binding site can be distinguished. In the first, the substrate is fully engaged and the nucleophilic nitrogen held in place by hydrogen bonds. In the second, the indole ring is further away from the catalytic site, providing space for the ethylamide to change its conformation and engage in a variety of alternative interactions. This disengaged state is incompatible with methyl transfer, but likely to play a role during initial substrate capture, subsequent deprotonation of the amine and, following the reaction, as a first step towards product release. Our study also sheds a new light on the evolution of SAMdependent methyltransferases and particularly on their ability to adapt to novel substrates. Consistent with the idea that highly variable loop regions inserted into the core Rossmann fold play a crucial role in this process, we find that the SRL of PsiMoriginally a motif interacting with stem-loop structures in RNAdeveloped into a highly specialised lid structure that recognises the small-molecule substrate and sequesters it by closing off the binding site (Fig.). In contrast, the Rossmann fold proper, the substrate-binding pocket and the catalytic centre remained virtually unchanged in the course of evolution. Emergence of PsiM from m 6 A-methyltransferases appears to have been driven by substrate mimicry, ultimately resulting in a moderately efficient small-molecule dimethyltransferase. Although the presence of N247 in PsiM, instead of M274 as in METTL16, enables PsiM to catalyse dimethylation, the second methyl transfer remains less efficient than the first. Moreover, exchange of SAH for SAM requires release of the intermediate product, baeocystin, further hampering the second round of methylation and favouring accumulation of the monomethylated species. This may indicate that the enzyme is caught in local optimum, an "evolutionary cul-de-sac", as a consequence of the inherent limitations of its original scaffold. Consistent with this idea, small-molecule methyltransferases that are known to efficiently di-or trimethylate amines, such as the histidine methyltransferase EgtD, the N,N-8-demethyl-8-amino-D-riboflavin methyltransferase RosAand the phosphoethanolamine methyltransferase PfPMT, possess an altogether different active site architecture without an NPPF motif and are capable of exchanging the coenzyme while the substrate remains bound. With the notable exception of PsiM, the NPPF motif has only been found in N-methyltransferases acting on planar substrates where the target amine is part of a conjugated system. It thus seems plausible that the rigid geometry of the proline-rich sequence helps imposing sp 3 hybridisation, rather than sp 2 , while guiding the lone pair towards the methyl group of SAM. The fact that the amines in the psilocybin precursors norbaeocystin and baeocystin are not part of a conjugated system and, in principle, should not benefit from such a mechanism, is a further indication pointing at the evolutionary origins of PsiM. Reactive oligomers resulting from oxidation of the phenolic hydroxyl group of psilocin, the product of spontaneous dephosphorylation of psilocybin, precipitate proteins and cause intracellular damage. Hallucinogenic mushrooms protect themselves by means of another enzyme of the psilocybin biosynthetic pathway, the promiscuous kinase PsiK, which re-phosphorylates psilocin to psilocybin. Formation of the tertiary amine is advantageous to the fungus as it enhances intramolecular hydrogen bonding between the phenolic hydroxyl group of the dephosphorylated species and the ethylamine. This effect markedly slows down oxidation, enabling PsiK to keep pace with the dephosphorylation process. In the light of these considerations, PsiM's incapacity to perform a third methylation seems consistent with the absence of protective hydrogen bond formation in aeruginascin's dephosphorylated degradation product, 4-hydroxy-N,N,N-trimethyltryptamine. The METTL16 family of methyltransferases was recently discovered and shown to represent a novel class of epitranscriptomic "writer" proteins, which place regulatory mA marks on mRNA. METTL16 was also found to modify various kinds of non-coding RNA, including U6 snRNA. In humans, aberrant RNA modification patterns are frequently associated with disease, leading to significant interest in the emerging field of epitranscriptomics. PsiM and the methyltransferase domain of human METTL16 are closely related and retain 37% sequence identity. Consequently, our results are likely to provide direct insight into the catalytic mechanism of METTL16. While the latter could be crystallised in its apo-formand in complexes containing either SAHor RNA, cocrystals with both ligands remain elusive. Our structures of PsiM can be used as templates to model the corresponding ternary complexes of METTL16. Moreover, in view of the resemblance between the substrate-binding modes of the two enzymes, norbaeocystin and baeocystin may turn out to be effective lead compounds for the design of therapeutic METTL16 inhibitors, or indeed of novel general-purpose nucleotide analogues. Despite the remarkable level of similarity between PsiM and METTL16, several intriguing differences are also apparent. In the first place, the enzymatic activity of METTL16 is tightly controlled, consistent with its role in epitranscriptomic gene regulation. Residues Lys163 and Met167 of the so-called K-loop, which play essential roles in the regulatory mechanism, have not been conserved in PsiM (Supplementary Fig.). Furthermore, all METTL16 proteins carry variable and partially disordered C-terminal extensions, comprising a pair of structured "vertebrate conserved region" (VCR) domains that influence RNAbinding. The VCR domains were also reported to mediate dimerisation, although this has been contested more recently. Finally, METTL16 was shown to bind RNA before it recruits SAM, whereas our results indicate that PsiM must recruit its cofactor before it is able to interact with substrates. It is conceivable that initial binding of RNA to the METTL16 surface alonetriggers SAM recruitment. The presence of the coenzyme might then stabilise the active site and enable it to sequester the target nucleotide. Accurate three-dimensional protein structures are key assets in bioengineering, enabling rational modification of enzymes and their substrates. Inspection of the substrate-binding pocket of PsiM reveals that moderately sized substituents at positions 6 and 7 of the indole ring are likely to be tolerated by the wild-type enzyme. Substitutions at the remaining ring positions, on the other hand, are likely to require substantial remodelling of the active site through mutagenesis. The phosphate moiety seems especially important for substrate recognition and presumably cannot be deleted or moved to a different position on the ring without severely affecting binding affinity. These structure-based findings are confirmed by recent experiments involving alternative substrates in psilocybin-producing E. coli strains. As the relevance of psilocybin as a future medication against a growing number of common mental health conditions is becoming clear, we expect that the atomic-resolution structures of PsiM will constitute important tools in bioengineering efforts aimed at producing novel analogues with improved therapeutic properties.
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