Part:BBa_K3140003
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].
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.
A band consistent with expression of PsiM in cells induced with IPTG was observed on polyacrylamide gel electrophoresis (Fig. 3).
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%.
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.
Retention time (min) | Signal/noise ratio | Measured m/z | Formula | Ion identity |
---|---|---|---|---|
0.56 | 23.2 | 271.0817 | C12H15O7 | unknown |
1.16 | 12.4 | 257.0689 | C10H14N2O4P | norbaeocystin |
1.9 | 0.9 | 271.0844 | C11H16N2O4P | baeocystin |
2.75 | 5.2 | 177.1023 | C10H13N2O | hydroxytryptamine |
5.08 | 6.4 | 205.0972 | C11H13N2O2 | tryptophan |
5.82 | 6.4 | 161.1074 | C10H13N2 | tryptamine |
10.15 | 14.4 | 285.1335 | C14H21O6 | unknown |
10.15 | 14.4 | 285.1335 | C9H18N8OP | unknown |
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).
However, pUS387 sequencing data showed a significant deletion in PsiM. This truncated PsiM sequence appeared to be approximately 519 bp in length (Fig. 7).
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).
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
None |