Difference between revisions of "Part:BBa K3140003"

Revision as of 06:14, 15 October 2019

PsiM - Norbaeocystin methyltransferase from Psilocybe cubensis

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

NCBI: ASU62238.1
UniProt: P0DPA9
EC number: 2.1.1.345

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. 2), resulting in a 345 amino acid polypeptide, with a computed molecular weight of 38.2 kDa.

Fig. 2: 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. 3).

Fig. 3: 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

↑ 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).

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

[ExPASy-2] Artimo, P. et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 40, W597-603 (2012).

[1]

[2]

@@ Line 17: / Line 17: @@
 [[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]  ]]
-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.
+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. 2'''), resulting in a 345 amino acid polypeptide, with a computed molecular weight of 38.2 kDa.
-[[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.]]
+[[Image:T--Sydney_Australia--PsiM_pET28c_map.png|700px|frame|none|'''Fig. 2''': 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''').
+A band consistent with expression of PsiM in cells induced with IPTG was observed on polyacrylamide gel electrophoresis ('''Fig. 3''').
-[[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.]]
+[[Image:T--Sydney_Australia--PsiM_pET28c_SDS.png|700px|frame|none|'''Fig. 3''': 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>