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., 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.

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 and insoluble protein extract from uninduced and IPTG induced E. coli BL21(DE3)::pGro7 cells containing pET-28c(+):PsiM, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 200V for 40 minutes.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]