Part:BBa_K3140004

PsiH (codon optimised) - Cytochrome P450 monooxygenase from Psilocybe cubensis

PsiH (codon optimised) is a cytochrome P450 monooxygenase, which catalyses the conversion of tryptamine to 4-hydroxytryptamine.

• 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., 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.

PsiH (codon optimised) is an enzyme derived from Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. The coding sequence has been codon optimised for expression in Escherichia coli. It accepts tryptamine as a substrate to yield 4-hydroxytryptamine (Fig. 1). In a native state, PsiH is a 508 amino acid protein (57.5 kDa) with a theoretical pI of 5.94 calculated with the ExPASy ProtParam tool.

Fig. 1: Hydroxylation of tryptamine to 4-hydroxytryptamine, mediated by PsiH. The reaction relies on a hydrogen donor (C00030) and O₂, which results in the release of the oxidised donor (C00028) and H₂O as a by-product. Source: KEGG

Heterologous expression of PsiH has been achieved in a T7 induction system using pCW (MORE) transformed into Escherichia coli BL21(DE3), co-transformed with chaperone plasmid pGro7 (Fig. 3).

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]