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]  ]]
  
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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''').
+
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.]]
 
[[Image:T--Sydney_Australia--pUS382_map.png|700px|frame|none|'''Fig. 3''': pUS382 plasmid map, showing PsiH and the human cytochrome P450 reductase.]]
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[[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.]]
 
[[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.]]
  
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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<ref name="indigo">Gillam, E.M. & Guengerich, F.P. Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology. ''IUBMB Life'' '''52''', 271-7 (2001).</ref>, 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''').
+
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<ref name="indigo">Gillam, E.M. & Guengerich, F.P. Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology. ''IUBMB Life'' '''52''', 271-7 (2001).</ref>, 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.]]
 
[[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.]]
 +
 +
This result does not rule out the functionality of PsiH, as it is known that not all P450 enzymes catalyse the formation of indigo<ref name="indigo" />. In order to confirm activity of PsiH, we conducted LC/MS on whole cell cultures of pUS382 that were provided with the PsiH substrate, tryptamine.
 +
 +
{| class="wikitable"
 +
|+ ''Table 1'': Identified compounds in LC/MS of protein extract of ''E. coli'' BL21(DE3) co-transformed with pUS382, with the addition of tryptamine.
 +
|-
 +
! Retention time (min)
 +
! Signal/noise ratio
 +
! Measured m/z
 +
! Formula
 +
! Ion identity
 +
|-
 +
| 0.58
 +
| 5.6
 +
| 271.0817
 +
| C12H15O7
 +
| unknown
 +
|-
 +
| 5.46
 +
| 23
 +
| 161.1074
 +
| C10H13N2
 +
| tryptamine
 +
|-
 +
| 10.14
 +
| 0.8
 +
| 285.1335
 +
| C9H18N8OP
 +
| unknown
 +
|-
 +
| 10.14
 +
| 0.8
 +
| 285.1335
 +
| C14H21O6
 +
| unknown
 +
|}
 +
 +
As we could not identify the PsiH product 4-hydroxytryptamine in the LC/MS results, we could not confirm the activity of PsiH ''in vivo''.
  
 
<span class='h3bb'>'''Sequence and Features'''</span>
 
<span class='h3bb'>'''Sequence and Features'''</span>

Latest revision as of 08:44, 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.

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].

Fig. 1: Hydroxylation of tryptamine to 4-hydroxytryptamine, mediated by PsiH. The reaction relies on a hydrogen donor (C00030) and O2, which results in the release of the oxidised donor (C00028) and H2O as a by-product. Source: 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).

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.

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.

As indole, when hydroxylated, is converted into indigo pigment through autoxidation[3], we sought to test whether PsiH, like many cytochrome p450 enzymes[4], 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).

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.

This result does not rule out the functionality of PsiH, as it is known that not all P450 enzymes catalyse the formation of indigo[4]. In order to confirm activity of PsiH, we conducted LC/MS on whole cell cultures of pUS382 that were provided with the PsiH substrate, tryptamine.

Table 1: Identified compounds in LC/MS of protein extract of E. coli BL21(DE3) co-transformed with pUS382, with the addition of tryptamine.
Retention time (min) Signal/noise ratio Measured m/z Formula Ion identity
0.58 5.6 271.0817 C12H15O7 unknown
5.46 23 161.1074 C10H13N2 tryptamine
10.14 0.8 285.1335 C9H18N8OP unknown
10.14 0.8 285.1335 C14H21O6 unknown

As we could not identify the PsiH product 4-hydroxytryptamine in the LC/MS results, we could not confirm the activity of PsiH in vivo.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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).
  3. ↑ 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).
  4. ↑ 4.0 4.1 Gillam, E.M. & Guengerich, F.P. Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology. IUBMB Life 52, 271-7 (2001).