Difference between revisions of "Part:BBa K3140001"
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[[Image:T--Sydney_Australia--PsiH_KEGG_rxn.gif|frame|none|Fig. 1: Hydroxylation of tryptamine to 4-hydroxytryptamine, mediated by PsiH. The reaction relies on a hydrogen donor (C00030) and O<sub>2</sub>, which results in the release of the oxidised donor (C00028) and H<sub>2</sub>O as a by-product. Source: [https://www.genome.jp/dbget-bin/www_bget?reaction+R11912 KEGG] ]] | [[Image:T--Sydney_Australia--PsiH_KEGG_rxn.gif|frame|none|Fig. 1: Hydroxylation of tryptamine to 4-hydroxytryptamine, mediated by PsiH. The reaction relies on a hydrogen donor (C00030) and O<sub>2</sub>, which results in the release of the oxidised donor (C00028) and H<sub>2</sub>O as a by-product. Source: [https://www.genome.jp/dbget-bin/www_bget?reaction+R11912 KEGG] ]] | ||
| − | + | Heterologous expression of PsiH has been achieved in a IPTG-based induction system based on pCW-CYP26A. Following removal of the human cytochrome p450 2A6 from pCW-CYP26A to yield pUS381, PsiH was cloned into pUS381 to yield pUS382, which was then transformed into ''Escherichia coli'' '''BL21(DE3)'', co-transformed with chaperone plasmid pGro7 ('''Fig. 3'''). | |
| − | + | [[Image:T--Sydney_Australia--pUS382_map.png|700px|frame|none|'''Fig. 3''': pUS382 plasmid map, showing PsiH and the human cytochrome P450 reductase.]] | |
| + | A band consistent with expression of PsiH in cells induced with IPTG was observed on polyacrylamide gel electrophoresis ('''Fig. 4'''), however, these bands could not be confirmed by peptide mass fingerprinting. | ||
| + | [[Image:T--Sydney_Australia--pUS38123_gel.png|frame|none|'''Fig. 4''': Polyacrylamide gel electrophoresis image of soluble and insoluble protein extracts from uninduced and IPTG induced ''E. coli'' BL21(DE3)::pGro7 cells containing pUS381, pUS382, and pUS383, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 120V for 60 minutes.]] | ||
| − | <span class='h3bb'>Sequence and Features</span> | + | As indole, when hydroxylated, is converted into indigo pigment through autoxidation<ref name="indole">Banoglu, E., Jha, G.G. & King, R.S. Hepatic microsomal metabolism of indole to indoxyl, a precursor of indoxyl sulfate. ''Eur J Drug Metab Pharmacokinet'' '''26''', 235-40 (2001)</ref>, we sought to test whether PsiH, like many cytochrome p450 enzymes, could catalyse this reaction. We conducted an indole assay on pCW-CYP26A, pUS381, pUS382, and pUS383, but only observed the production of pigment in pCW-CYP26A ('''Fig. 5'''). |
| + | |||
| + | [[Image:T--Sydney_Australia--indole.jpg|frame|none|'''Fig. 5''': IPTG-induced cultures of pCW-CYP26A, pUS381, pUS382, and pUS383 following completion of indole assay. Indigo production is only observed in the pCW-CYP26A culture.]] | ||
| + | |||
| + | <span class='h3bb'>'''Sequence and Features'''</span> | ||
<partinfo>BBa_K3140001 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3140001 SequenceAndFeatures</partinfo> | ||
Revision as of 08:17, 20 October 2019
PsiH - Cytochrome P450 monooxygenase from Psilocybe cubensis
PsiH is a cytochrome P450 monooxygenase that catalyses the conversion of tryptamine to 4-hydroxytryptamine.
- NCBI: ASU62246.1
- UniProt: P0DPA7
- EC number: 1.14.99.59
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.
PsiH is a native enzyme obtained from Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. It accepts tryptamine as a substrate to yield 4-hydroxytryptamine (Fig. 1). In a native state, PsiH is a 508 amino acid protein (57.5 kDa) with a theoretical pI of 5.94 calculated with the ExPASy ProtParam tool[2].
Heterologous expression of PsiH has been achieved in a IPTG-based induction system based on pCW-CYP26A. Following removal of the human cytochrome p450 2A6 from pCW-CYP26A to yield pUS381, PsiH was cloned into pUS381 to yield pUS382, which was then transformed into Escherichia coli BL21(DE3), co-transformed with chaperone plasmid pGro7 ('Fig. 3).
A band consistent with expression of PsiH in cells induced with IPTG was observed on polyacrylamide gel electrophoresis (Fig. 4), however, these bands could not be confirmed by peptide mass fingerprinting.
As indole, when hydroxylated, is converted into indigo pigment through autoxidation[3], we sought to test whether PsiH, like many cytochrome p450 enzymes, could catalyse this reaction. We conducted an indole assay on pCW-CYP26A, pUS381, pUS382, and pUS383, but only observed the production of pigment in pCW-CYP26A (Fig. 5).
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]
References
- ↑ Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. Angew Chem Int Ed Engl 56, 12352-12355 (2017).
- ↑ Artimo, P. et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40, W597-603 (2012).
- ↑ Banoglu, E., Jha, G.G. & King, R.S. Hepatic microsomal metabolism of indole to indoxyl, a precursor of indoxyl sulfate. Eur J Drug Metab Pharmacokinet 26, 235-40 (2001)