Difference between revisions of "Part:BBa K3140000"
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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="LoC">Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. ''Angew Chem Int Ed Engl'' '''56''', 12352-12355 (2017).</ref>.  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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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>.  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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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.
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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<ref name="ExPASy">Artimo, P. et al. ExPASy: SIB bioinformatics resource portal. ''Nucleic Acids Res'' '''40''', W597-603 (2012).</ref>.
  
 
[[Image:T--Sydney_Australia--PsiD_KEGG_rxn1.gif|frame|none|'''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_rxn1.gif|frame|none|'''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]  ]]
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[[Image:T--Sydney_Australia--PsiD_pET28c_SDS.png|700px|frame|none|'''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.]]
 
[[Image:T--Sydney_Australia--PsiD_pET28c_SDS.png|700px|frame|none|'''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.]]
  
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===References===
 
  
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{{reflist}}
 
  
 
# Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. ''Angew Chem Int Ed Engl'' '''56''', 12352-12355 (2017).
 
# Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. ''Angew Chem Int Ed Engl'' '''56''', 12352-12355 (2017).
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# Artimo, P. et al. ExPASy: SIB bioinformatics resource portal. ''Nucleic Acids Res'' '''40''', W597-603 (2012).
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#  
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3140000 parameters</partinfo>
 
<partinfo>BBa_K3140000 parameters</partinfo>
 
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===References===

Revision as of 02:51, 15 October 2019


PsiD - Tryptophan decarboxylase from Psilocybe cubensis

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

Usage and Biology

The mechanism of psilocybin biosynthesis in Psilocybe sp. was recently elucidated by Fricke et al.[1]. 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 byproduct. Source: KEGG
Fig. 2: Decarboxylation of 4-hydroxy-L-tryptophan to 4-hydroxytryptamine, mediated by PsiD. CO2 is released as a byproduct. 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.


  1. Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. Angew Chem Int Ed Engl 56, 12352-12355 (2017).

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).