Difference between revisions of "Part:BBa K3140004"
 
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===Usage and Biology===
 
===Usage and Biology===
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The mechanism of psilocybin biosynthesis in ''Psilocybe'' sp. was recently elucidated by Fricke ''et al''.<ref name="Fricke">Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. ''Angew Chem Int Ed Engl'' '''56''', 12352-12355 (2017).</ref>, 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.
 
The mechanism of psilocybin biosynthesis in ''Psilocybe'' sp. was recently elucidated by Fricke ''et al''.<ref name="Fricke">Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. ''Angew Chem Int Ed Engl'' '''56''', 12352-12355 (2017).</ref>, 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.
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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 codon-optimised 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, codon-optimised PsiH was cloned into pUS381 to yield pUS383, which was then transformed into ''Escherichia coli'' BL21(DE3), co-transformed with chaperone plasmid pGro7 ('''Fig. 3''').
  
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Heterologous expression of PsiH has been achieved in a '''T7 induction system''' using '''pCW (MORE)'''  transformed into ''Escherichia coli'' '''BL21(DE3)''', co-transformed with chaperone plasmid pGro7 ('''Fig. 3''').
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[[Image:T--Sydney_Australia--pUS383_map.png|700px|frame|none|'''Fig. 3''': pUS383 plasmid map, showing codon-optimised PsiH and the human cytochrome P450 reductase.]]
  
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A band consistent with expression of codon-optimised 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.
  
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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.]]
  
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<span class='h3bb'>Sequence and Features</span>
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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 codon-optimised 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''').
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[[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.]]
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This result does not rule out the functionality of codon-optimised 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 codon-optimised PsiH substrate, tryptamine.
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{| class="wikitable"
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|+ ''Table 1'': Identified compounds in LC/MS of protein extract of ''E. coli'' BL21(DE3) co-transformed with pUS383, with the addition of tryptamine.
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|-
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! Retention time (min)
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! Signal/noise ratio
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! Measured m/z
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! Formula
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! Ion identity
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|-
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| 0.57
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| 5.8
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| 271.0817
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| C12H15O7
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| unknown
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|-
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| 2.68
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| 6.8
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| 177.1023
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| C10H13N2O
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| hydroxytryptamine
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|-
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| 5.47
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| 26.5
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| 161.1074
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| C10H13N2
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| tryptamine
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|-
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| 10.14
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| 5.3
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| 285.1335
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| C14H21O6
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| unknown
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|-
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| 10.14
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| 5.3
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| 285.1335
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| C9H18N8OP
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| unknown
 +
|}
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Although we identified a hydroxytryptamine in the LC/MS results ('''Table 1'''), we could not confirm that this was due to the activity of PsiH, as we identified hydroxytryptamine in our tryptamine standards ('''Table 2''').
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{| class="wikitable"
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|+ ''Table 2'': Identified compounds in LC/MS of a 1 mM tryptamine standard
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|-
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| 2.62
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| 3.3
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| 177.1023
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| C10H13N2O
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| hydroxytryptamine
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|-
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| 4.24
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| 38.2
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| 161.1075
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| C10H13N2
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| tryptamine
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|}
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We could not confirm the isomeric form of the hydroxytryptamine in this sample, but we speculate that it may be present in the standard due to autoxidation of tryptamine.
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<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K3140001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3140001 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K3140001 parameters</partinfo>
 
<partinfo>BBa_K3140001 parameters</partinfo>
 
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==References==

Latest revision as of 08:58, 20 October 2019


PsiH (codon optimised) - Cytochrome P450 monooxygenase from Psilocybe cubensis

PsiH (codon optimised) is a cytochrome P450 monooxygenase, which 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 (codon optimised) is an enzyme derived from Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. The coding sequence has been codon optimised for expression in Escherichia coli. 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 codon-optimised 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, codon-optimised PsiH was cloned into pUS381 to yield pUS383, which was then transformed into Escherichia coli BL21(DE3), co-transformed with chaperone plasmid pGro7 (Fig. 3).

Fig. 3: pUS383 plasmid map, showing codon-optimised PsiH and the human cytochrome P450 reductase.

A band consistent with expression of codon-optimised 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 codon-optimised 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 codon-optimised 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 codon-optimised PsiH substrate, tryptamine.

Table 1: Identified compounds in LC/MS of protein extract of E. coli BL21(DE3) co-transformed with pUS383, with the addition of tryptamine.
Retention time (min) Signal/noise ratio Measured m/z Formula Ion identity
0.57 5.8 271.0817 C12H15O7 unknown
2.68 6.8 177.1023 C10H13N2O hydroxytryptamine
5.47 26.5 161.1074 C10H13N2 tryptamine
10.14 5.3 285.1335 C14H21O6 unknown
10.14 5.3 285.1335 C9H18N8OP unknown

Although we identified a hydroxytryptamine in the LC/MS results (Table 1), we could not confirm that this was due to the activity of PsiH, as we identified hydroxytryptamine in our tryptamine standards (Table 2).

Table 2: Identified compounds in LC/MS of a 1 mM tryptamine standard
2.62 3.3 177.1023 C10H13N2O hydroxytryptamine
4.24 38.2 161.1075 C10H13N2 tryptamine

We could not confirm the isomeric form of the hydroxytryptamine in this sample, but we speculate that it may be present in the standard due to autoxidation of tryptamine.

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).