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 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).

Table 1: Identified compounds in LC/MS of protein extract of E. coli BL21(DE3) co-transformed with pET-28c(+):PsiK and pGro7, with the addition of 4-hydroxytryptamine.

Retention time (min)	Signal/noise ratio	Measured m/z	Formula	Ion identity
1.16	0.9	257.0687	C10H14N2O4P	norbaeocystin
2.63	20.7	177.1024	C10H13N2O	hydroxytryptamine
3.46	2.4	205.0972	C11H13N2O2	tryptophan
10.14	5.1	285.1336	C9H18N8OP	unknown
10.14	5.1	285.1336	C14H21O6	unknown

Table 2: Identified compounds in LC/MS of protein extract of E. coli BL21(DE3) co-transformed with pET-28c(+):PsiK and pGro7, without the addition of 4-hydroxytryptamine..

Retention time (min)	Signal/noise ratio	Measured m/z	Formula	Ion identity
10.16	11.3	285.1336	C14H21O6	unknown
10.16	11.3	285.1336	C9H18N8OP	unknown

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.

Table 3: Identified compounds in LC/MS of protein extract of E. coli DH5α co-transformed with pUS387 and pGro7, with the addition of 4-hydroxytryptamine.

Retention time (min)	Signal/noise ratio	Measured m/z	Formula	Ion identity
0.56	23.2	271.0817	C12H15O7	unknown
1.16	12.4	257.0689	C10H14N2O4P	norbaeocystin
1.9	0.9	271.0844	C11H16N2O4P	baeocystin
2.75	5.2	177.1023	C10H13N2O	hydroxytryptamine
5.08	6.4	205.0972	C11H13N2O2	tryptophan
5.82	6.4	161.1074	C10H13N2	tryptamine
10.15	14.4	285.1335	C14H21O6	unknown
10.15	14.4	285.1335	C9H18N8OP	unknown

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

Assembly Compatibility:

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  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
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  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

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