Part:BBa_K3140000

PsiD - Tryptophan decarboxylase from Psilocybe cubensis

PsiD is a tryptophan decarboxylase that catalyses the conversion of L-tryptophan to tryptamine.

• NCBI: ASU62239.1
• UniProt: P0DPA6
• EC number: 4.1.1.105

Usage and Biology

The mechanism of psilocybin biosynthesis in Psilocybe sp. was recently elucidated by Fricke et al. (2017). 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.

PsiD is a native enzyme obtained from the Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. It accepts both L-tryptophan and 4-hydroxy-L-tryptophan as substrates, producing tryptamine (Fig. 1) and 4-hydroxytryptamine (Fig. 2), respectively. In a native state, PsiD is a 439 amino acid protein (49.6 kDa) with a theoretical pI of 5.44 calculated with the ExPASy ProtParam tool.

Fig. 1: Decarboxylation of L-tryptophan to tryptamine, mediated by PsiD. CO₂ is released as a byproduct. Source: KEGG

Fig. 2: Decarboxylation of 4-hydroxy-L-tryptophan to 4-hydroxytryptamine, mediated by PsiD. CO₂ is released as a byproduct. Source: KEGG

Heterologous expression of PsiD has been achieved in a T7 induction system using pET-28c(+) transformed into Escherichia coli BL21(DE3), co-transformed with chaperone plasmid pGro7 (Fig. 3), resulting in a 475 amino acid polypeptide, with a computed molecular weight of 53.6 kDa.

Fig. 3: pET-28c(+):PsiD 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 PsiD in cells induced with IPTG was observed on polyacrylamide gel electrophoresis (Fig. 4).

Fig. 4: Polyacrylamide gel electrophoresis image of soluble and insoluble protein extract from uninduced and IPTG induced E. coli BL21(DE3)::pGro7 cells containing pET-28c(+):PsiD, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 200V for 40 minutes.

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).

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]