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PsiD - Tryptophan decarboxylase from Psilocybe cubensis

PsiD is a tryptophan decarboxylase that catalyses the conversion of L-tryptophan to tryptamine.

• 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.^[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.

PsiD is a native enzyme obtained from the Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. It accepts both L-tryptophan and 4-hydroxy-L-tryptophan as substrates, producing tryptamine (Fig. 1) and 4-hydroxytryptamine (Fig. 2), respectively. In a native state, PsiD is a 439 amino acid protein (49.6 kDa) with a theoretical pI of 5.44 calculated with the ExPASy ProtParam tool^[2].

Fig. 1: Decarboxylation of L-tryptophan to tryptamine, mediated by PsiD. CO₂ is released as a by-product. Source: KEGG

Fig. 2: Decarboxylation of 4-hydroxy-L-tryptophan to 4-hydroxytryptamine, mediated by PsiD. CO₂ is released as a by-product. Source: KEGG

Heterologous expression of PsiD 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 475 amino acid polypeptide, with a computed molecular weight of 53.6 kDa.

Fig. 3: pET-28c(+):PsiD 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 PsiD 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(+):PsiD, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 200V for 40 minutes.

Activity of PsiD was confirmed with LC/MS. Protein extract from E. coli BL21(DE3) cells co-transformed with pET-28c(+):PsiD and pGro7 was subject to LC/MS, both with and without the addition of the PsiD substrate, tryptophan. The PsiD product, tryptamine, was only identified in the sample to which tryptophan was added, confirming the activity of PsiD in vitro (Fig. 5).

Fig. 5: Mass spectra obtained LC/MS of protein extract of E. coli BL21(DE3) co-transformed with pET-28c(+):PsiD and pGro7, with the addition of tryptophan. A peak at m/z = 161.10732 with chemical formula C10H13N2 was identified, matching PsiD product tryptamine.

The presence of tryptophan in sample to which tryptophan was added may indicate autoxidation of tryptamine, as this compound was also observed in the mass spectrum of a 1 mM tryptamine standard (Table 1). However, neither hydroxytryptamine nor tryptamine was not observed in the sample to which tryptophan was not added (Table 2).

===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).