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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.^[1], 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^[2].

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 protein extract from uninduced and IPTG induced E. coli BL21(DE3)::pGro7 cells containing pET-28c(+):PsiD, pET-28c(+):PsiK, and pET-28c(+):PsiM, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 120V for 60 minutes.

Activity of PsiK was confirmed with LC/MS. Protein extract from E. coli BL21(DE3) cells co-transformed with pET-28c(+):PsiK and pGro7 was subject to LC/MS, both with and without the addition of the PsiK substrate, 4-hydroxytryptamine. The PsiK product, norbaeocystin, was only identified in the sample to which 4-hydroxytryptamine was added, confirming the activity of PsiK in vitro (Table 1).

While norbaeocystin was observed in the sample to which 4-hydroxytryptamine was added (Table 1), it was not observed when 4-hydroxytryptamine was absent (Table 2).

In vivo expression of PsiK was also confirmed. PsiD, PsiK, and PsiM were cloned into a pUS250 backbone as a gene cluster using Golden Gate cloning, yielding pUS387 (Fig. 5). Expression in pUS387 is driven by a class 1 integron gene cassette Pc promoter controlled by a cumate induction system. E. coli DH5α cells co-transformed with pUS387 and pGro7 were cultured in terrific broth (TB) supplemented with 4-hydroxytryptamine. Whole cell culture was subject to LC/MS.

Fig. 5: pUS387 plasmid map, showing PsiD, PsiK, and PsiM genes organised in a cluster, driven by a class 1 integron gene cassette Pc promoter, flanked by CuO, a cumate repressor binding operator.

The in vivo activity of PsiK is confirmed by observation of PsiK product norbaeocystin (Fig. 6).

Fig. 6: Mass spectra obtained LC/MS of protein extract of E. coli DH5α co-transformed with pUS387 and pGro7, with the addition of 4-hydroxytryptamine. A peak at m/z = 257.06857 with chemical formula C10H14N2O4P was identified, matching PsiK product norbaeocystin.

Finally, confirmation of expression in pET-28c(+):PsiK was gained by gained by peptide mass fingerprinting (Fig. 7), which matched to PsiM with an identity score of 49 (0.00011 expected) and protein sequence coverage of 49%.

Fig. 7: Polyacrylamide gel containing soluble cell lysate profiles from pET-28c(+):PsiM and pET-28c(+):PsiK cultures believed to be expressing soluble PsiM and PsiK, stained with Coomassie Blue. The bands highlighted in red were cut, destained with 40% acetonitrile/60% 20mM ammonium bicarbonate. Cut bands were then dehydrated, and then rehydrated with 12ng/ul porcine trypsin at 4°C for 30min. Excess trypsin was removed and 10 μl of 20mM ammonium bicarbonate was added. Following overnight incubation at 37°C. Peptide masses were determined using mass spectroscopy, and samples were identified using a Mascot peptide mass fingerprint search.

Given these results, we conclude that we have successfully expressed the 4-hydroxytryptamine kinase decarboxylase PsiK from Psilocybe cubensis in Escherichia coli both in vivo and in vitro.

Sequence and Features

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).