Part:BBa_K3140002

PsiK - 4-hydroxytryptamine kinase from Psilocybe cubensis

PsiK is a 4-hydroxytryptamine kinase, which catalyses the conversion of 4-hydroxytryptamine to norbaeocystin.

• NCBI: ASU62237.1
• UniProt: P0DPA8
• EC number: 2.7.1.222

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.

PsiK is a native enzyme obtained from Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. It accepts both 4-hydroxytryptamine and psilocin as substrates to yield norbaeocystin (Fig. 1) and psilocybin (Fig. 2), respectively. In a native state, PsiK is a 362 amino acid protein (40.4 kDa) with a theoretical pI of 5.26 calculated with the ExPASy ProtParam tool.

Fig. 1: Phosphorylation of 4-hydroxytryptamine to norbaeocystin, mediated by PsiK. The reaction is dependant upon ATP, and ADP is released as a by-product. Source: KEGG

Fig. 2: Phosphorylation of psilocin to psilocybin, mediated by PsiK. The reaction is dependant upon ATP, and ADP is released as a by-product. Source: KEGG

Heterologous expression of PsiK 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 397 amino acid polypeptide, with a computed molecular weight of 44.2 kDa.

Fig. 3: pET-28c(+):PsiK 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 PsiK 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(+):PsiK, 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
  INCOMPATIBLE WITH RFC[1000]

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