Difference between revisions of "Part:BBa K3140000"

Revision as of 00:51, 15 October 2019

PsiD - Tryptophan decarboxylase from Psilocybe cubensis

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

NCBI: ASU62239.1
UniProt: P0DPA6
EC number: 4.1.1.105

Usage and Biology

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 and 4-hydroxytryptamine, respectively. 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. 4).

Heterologous expression of PsiD in pET-28c(+) yields a 475 amino acid polypeptide, with a computed molecular weight of 53550.71 Da.

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

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.

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.

ADD:

- SDS-PAGE IMAGE - LC/MS - pUS250 and pET-28c(+)

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]

@@ Line 11: / Line 11: @@
 ===Usage and Biology===
-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 and 4-hydroxytryptamine, respectively. 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''').
+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 and 4-hydroxytryptamine, respectively. 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. 4''').
 Heterologous expression of PsiD in pET-28c(+) yields a 475 amino acid polypeptide, with a computed molecular weight of 53550.71 Da.
@@ Line 19: / Line 19: @@
 [[Image:T--Sydney_Australia--PsiD_KEGG_rxn2.gif|thumb|900px|Fig. 2: Decarboxylation of 4-hydroxy-L-tryptophan to 4-hydroxytryptamine, mediated by PsiD. CO<sub>2</sub> is released as a byproduct. Source: [https://www.genome.jp/dbget-bin/www_bget?reaction+R11932 KEGG]  ]]
-[[Image:T--Sydney_Australia--PsiD_pET28c_SDS.png|thumb|900px|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(+):PsiD, run on an Mini-PROTEAN® TGX Stain-Free™ precast gel (Bio-Rad) at 200V for 40 minutes.]]
+[[Image:T--Sydney_Australia--PsiD_pET28c_map.png|thumb|900px|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.]]
+[[Image:T--Sydney_Australia--PsiD_pET28c_SDS.png|thumb|900px|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.]]