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PsiM - Norbaeocystin methyltransferase from Psilocybe cubensis

PsiM is a norbaeocystin methyltransferase, which catalyses the conversion of norbaeocystin to psilocybin.

• NCBI: ASU62238.1
• UniProt: P0DPA9
• EC number: 2.1.1.345

Usage and Biology

The mechanism of psilocybin biosynthesis in Psilocybe sp. was recently elucidated by Fricke et al.^[1], demonstrating that L-tryptophan proceeds through decarboxylation (mediated by PsiD), hydroxylation (mediated by PsiH), phosphorylation (mediated by PsiK), and finally N,N-dimethylation (mediated by PsiM) to yield psilocybin.

PsiM is a native enzyme obtained from Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. It accepts norbaeocystin as a substrate to yield psilocybin through N,N-dimethylation (Fig. 1). In a native state, PsiM is a 309 amino acid protein (34.4 kDa) with a theoretical pI of 4.96 calculated with the ExPASy ProtParam tool^[2].

Fig. 1: N,N-dimethylation of norbaeocystin to psilocybin, mediated by PsiM. Intermediates not shown. Two S-adenosyl-L-methionine moieties are consumed, releasing two S-sdenosyl-L-homocysteine moieties as a by-product. Source: KEGG

Heterologous expression of PsiM has been achieved in a T7 induction system using pET-28c(+) transformed into Escherichia coli BL21(DE3), co-transformed with chaperone plasmid pGro7 (Fig. 2), resulting in a 345 amino acid polypeptide, with a computed molecular weight of 38.2 kDa.

Fig. 2: pET-28c(+):PsiM plasmid map, showing C-terminal histidine tag, and T7 promoter under the control of the lac operator. Translated peptide is shown as the thin lime green arrow.

A band consistent with expression of PsiM in cells induced with IPTG was observed on polyacrylamide gel electrophoresis (Fig. 3).

Fig. 3: Polyacrylamide gel electrophoresis image of soluble protein extract from uninduced and IPTG induced E. coli BL21(DE3)::pGro7 cells containing pET-28c(+):PsiD, pET-28c(+):PsiK, and pET-28c(+):PsiM, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 120V for 60 minutes.

Further confirmation was gained by peptide mass fingerprinting (Fig. 4), which matched to PsiM with an identity score of 57 (0.00023 expected) and protein sequence coverage of 50%.

Fig. 4: Polyacrylamide gel containing soluble cell lysate profiles from pET-28c(+):PsiM and pET-28c(+):PsiK cultures believed to be expressing soluble PsiM and PsiK, stained with Coomassie Blue. The bands highlighted in red were cut, destained with 40% acetonitrile/60% 20mM ammonium bicarbonate. Cut bands were then dehydrated, and then rehydrated with 12ng/ul porcine trypsin at 4°C for 30min. Excess trypsin was removed and 10 μl of 20mM ammonium bicarbonate was added. Following overnight incubation at 37°C. Peptide masses were determined using mass spectroscopy, and samples were identified using a Mascot peptide mass fingerprint search.

However, in vivo activity of PsiM could not be confirmed by LC/MS. PsiD, PsiK, and PsiM were cloned into a pUS250 backbone as a gene cluster using Golden Gate cloning, yielding pUS387 (Fig. 5). Expression in pUS387 is driven by a class 1 integron gene cassette Pc promoter controlled by a cumate induction system. E. coli DH5α cells co-transformed with pUS387 and pGro7 were cultured in terrific broth (TB) supplemented with 4-hydroxytryptamine. Whole cell culture was subject to LC/MS.

Fig. 5: pUS387 plasmid map, showing PsiD, PsiK, and PsiM genes organised in a cluster, driven by a class 1 integron gene cassette Pc promoter, flanked by CuO, a cumate repressor binding operator.

While in vitro activity of PsiM could not be conducted due to the lack of availability of reagent norbaeocystin, the in vivo activity of PsiM appeared to be confirmed by observation of PsiM product baeocystin (Fig. 6).

Fig. 6: Mass spectra obtained LC/MS of protein extract of E. coli DH5α co-transformed with pUS387 and pGro7, with the addition of 4-hydroxytryptamine. A peak at m/z = 271.08422 with chemical formula C10H14N2O4P was identified, matching PsiK product norbaeocystin.

However, pUS387 sequencing data showed a significant deletion in PsiM. This truncated PsiM sequence appeared to be approximately 519 bp in length (Fig. 7).

Fig. 7: pUS387 sequence map, showing poor alignment to sequence data obtained from Sanger sequencing.

This deletion was by a diagnostic restriction digest of pUS387, which showed a degraded band of approximately 500 bp, corresponding to the expected size of the truncated PsiM (Fig. 8).

Fig. 8: Double restriction digestion of pUS250 and pUS387 clones (14 and 16). Constructs were digested for 30 seconds using a 900 W microwave alongside a no template control using HindIII-HF and EcoRI-HF restriction enzymes. Products were loaded onto a 1% Agarose, 40mL TAE gel prestained with Gel Green. The gel was run for 40 minutes at 100V before being imaged under UV transillumination.

Given these results, we conclude that we have successfully expressed the norbaeocystin methyltransferase PsiM from Psilocybe cubensis in Escherichia coli in pET-28c(+):PsiM, but we could not confirm activity either in vitro or in vivo. Presence of baeocystin in pUS387 cell cultures may be due to an endogenous methyltransferase acting upon norbaeocystin.

Sequence and Features

References

1. ↑ Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. Angew Chem Int Ed Engl 56, 12352-12355 (2017).
2. ↑ Artimo, P. et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40, W597-603 (2012).