Difference between revisions of "Part:BBa K3140000"
| Line 19: | Line 19: | ||
A band consistent with expression of PsiD in cells induced with IPTG was observed on polyacrylamide gel electrophoresis ('''Fig. 4'''). | A band consistent with expression of PsiD in cells induced with IPTG was observed on polyacrylamide gel electrophoresis ('''Fig. 4'''). | ||
| − | [[Image:T--Sydney_Australia--PsiD_KEGG_rxn1.gif| | + | [[Image:T--Sydney_Australia--PsiD_KEGG_rxn1.gif|400px|'''Fig. 1''': Decarboxylation of L-tryptophan to tryptamine, mediated by PsiD. CO<sub>2</sub> is released as a byproduct. Source: [https://www.genome.jp/dbget-bin/www_bget?reaction+R00685 KEGG] ]] |
| − | [[Image:T--Sydney_Australia--PsiD_KEGG_rxn2.gif| | + | [[Image:T--Sydney_Australia--PsiD_KEGG_rxn2.gif|400px|'''Fig. 2''': Decarboxylation of 4-hydroxy-L-tryptophan to 4-hydroxytryptamine, mediated by PsiD. CO<sub>2</sub> is released as a byproduct. Source: [https://www.genome.jp/dbget-bin/www_bget?reaction+R11932 KEGG] ]] |
| − | [[Image:T--Sydney_Australia--PsiD_pET28c_map.png| | + | [[Image:T--Sydney_Australia--PsiD_pET28c_map.png|900px|'''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.]] |
| − | [[Image:T--Sydney_Australia--PsiD_pET28c_SDS.png| | + | [[Image:T--Sydney_Australia--PsiD_pET28c_SDS.png|900px|'''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.]] |
===References=== | ===References=== | ||
Revision as of 02:28, 15 October 2019
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. (2017). 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.
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.
A band consistent with expression of PsiD in cells induced with IPTG was observed on polyacrylamide gel electrophoresis (Fig. 4).
References
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]