Part:BBa_K3140001

PsiH - Cytochrome P450 monooxygenase from Psilocybe cubensis

PsiH is a cytochrome P450 monooxygenase that catalyses the conversion of tryptamine to 4-hydroxytryptamine.

• NCBI: ASU62246.1
• UniProt: P0DPA7
• EC number: 1.14.99.59

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 is a native enzyme obtained from Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. 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 IPTG-based induction system based on pCW-CYP26A. Following removal of the human cytochrome p450 2A6 from pCW-CYP26A to yield pUS381, PsiH was cloned into pUS381 to yield pUS382, which was then transformed into Escherichia coli BL21(DE3), co-transformed with chaperone plasmid pGro7 (Fig. 3).

Fig. 3: pUS382 plasmid map, showing PsiH and the human cytochrome P450 reductase.

A band consistent with expression of PsiH in cells induced with IPTG was observed on polyacrylamide gel electrophoresis (Fig. 4), however, these bands could not be confirmed by peptide mass fingerprinting.

Fig. 4: Polyacrylamide gel electrophoresis image of soluble and insoluble protein extracts from uninduced and IPTG induced E. coli BL21(DE3)::pGro7 cells containing pUS381, pUS382, and pUS383, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 120V for 60 minutes.

As indole, when hydroxylated, is converted into indigo pigment through autoxidation, we sought to test whether PsiH, like many cytochrome p450 enzymes, could catalyse this reaction. We conducted an indole assay on pCW-CYP26A, pUS381, pUS382, and pUS383, but only observed the production of pigment in pCW-CYP26A (Fig. 5).

Fig. 5: IPTG-induced cultures of pCW-CYP26A, pUS381, pUS382, and pUS383 following completion of indole assay. Indigo production is only observed in the pCW-CYP26A culture.

This result does not rule out the functionality of PsiH, as it is known that not all P450 enzymes catalyse the formation of indigo. In order to confirm activity of PsiH, we conducted LC/MS on whole cell cultures of pUS382 that were provided with the PsiH substrate, tryptamine.

Table 1: Identified compounds in LC/MS of protein extract of E. coli BL21(DE3) co-transformed with pUS382, with the addition of tryptamine.

Retention time (min)	Signal/noise ratio	Measured m/z	Formula	Ion identity
0.58	5.6	271.0817	C12H15O7	unknown
5.46	23	161.1074	C10H13N2	tryptamine
10.14	0.8	285.1335	C9H18N8OP	unknown
10.14	0.8	285.1335	C14H21O6	unknown

As we could not identify the PsiH product 4-hydroxytryptamine in the LC/MS results, we could not confirm the activity of PsiH in vivo.

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]