Difference between revisions of "Part:BBa K3140003"
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===Usage and Biology===
 
===Usage and Biology===
  
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PsiM is a native enzyme obtained from ''Psilocybe cubensis'', which is involved in the metabolic biosynthesis of psilocybin from tryptophan. It accepts norbaeocystin as a substrate to yield psilocybin through N,N-dimethylation. Heterologous expression of PsiM has been achieved in ''Escherichia coli'' <add strain> in a <expression system>.
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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.
  
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[[Image:T--Sydney_Australia--PsiM_KEGG_rxn.gif|thumb|900px|Fig. 1: N,N-dimethylation of norbaeocystin to psilocybin, mediated by PsiM. Intermediates not shown. Two ''S''-adenosyl-L-methionine moieties are consumed, releasing two ''S''-sdenosyl-L-homocysteine moieties as a by-product. Source: [https://www.genome.jp/dbget-bin/www_bget?reaction+R11934 KEGG]  ]]
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PsiM is a native enzyme obtained from ''Psilocybe cubensis'', which is involved in the metabolic biosynthesis of psilocybin from tryptophan. It accepts norbaeocystin as a substrate to yield psilocybin through N,N-dimethylation ('''Fig. 1'''). In a native state, PsiM is a 309 amino acid protein (34.4 kDa) with a theoretical pI of 4.96 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>.
  
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[[Image:T--Sydney_Australia--PsiM_KEGG_rxn.gif|frame|none|Fig. 1: N,N-dimethylation of norbaeocystin to psilocybin, mediated by PsiM. Intermediates not shown. Two ''S''-adenosyl-L-methionine moieties are consumed, releasing two ''S''-sdenosyl-L-homocysteine moieties as a by-product. Source: [https://www.genome.jp/dbget-bin/www_bget?reaction+R11934 KEGG]  ]]
  
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Heterologous expression of PsiM 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 345 amino acid polypeptide, with a computed molecular weight of 38.2 kDa.
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-SDS-PAGE - pCW - LC/MAS
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[[Image:T--Sydney_Australia--PsiM_pET28c_map.png|700px|frame|none|'''Fig. 3''': pET-28c(+):PsiM 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.]]
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A band consistent with expression of PsiM in cells induced with IPTG was observed on polyacrylamide gel electrophoresis ('''Fig. 4''').
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[[Image:T--Sydney_Australia--PsiM_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(+):PsiM, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 200V for 40 minutes.]]
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3140003 parameters</partinfo>
 
<partinfo>BBa_K3140003 parameters</partinfo>
 
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===References===

Revision as of 06:00, 15 October 2019


PsiM - Norbaeocystin methyltransferase from Psilocybe cubensis

PsiM is a norbaeocystin methyltransferase, which catalyses the conversion of norbaeocystin to psilocybin.

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.

PsiM is a native enzyme obtained from Psilocybe cubensis, which is involved in the metabolic biosynthesis of psilocybin from tryptophan. It accepts norbaeocystin as a substrate to yield psilocybin through N,N-dimethylation (Fig. 1). In a native state, PsiM is a 309 amino acid protein (34.4 kDa) with a theoretical pI of 4.96 calculated with the ExPASy ProtParam tool[2].

Fig. 1: N,N-dimethylation of norbaeocystin to psilocybin, mediated by PsiM. Intermediates not shown. Two S-adenosyl-L-methionine moieties are consumed, releasing two S-sdenosyl-L-homocysteine moieties as a by-product. Source: KEGG

Heterologous expression of PsiM 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 345 amino acid polypeptide, with a computed molecular weight of 38.2 kDa.

Fig. 3: pET-28c(+):PsiM 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 PsiM 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(+):PsiM, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 200V for 40 minutes.

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).