Part:BBa_K2271017
VioE
This part is the VioE enzyme of the violacein pathway and encoded by the VioABCDE operon. VioE catalyses the reaction from IPA imine dimer into Protodeoxyviolacein acid. It is naturally produced in gram-negative bacteria like chrombacterium violaceum and has numerous biological advantages. According to the given features in the sequence the part is optimized for Yeast (Promotor, Terminator), whereas the coding sequence is not. In the course of our project we did also work with VioA which is available as a biobrick as well (Part:BBa_K2271014).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal suffix found in sequence at 1561
Illegal BglII site found at 880 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1916
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Protein: 2-imino-3-(indol-3-yl)propanoate dimerase
The synthesis of Violacein requires five enzymes encoded by the VioABCDE operon. VioA, a Flavin-dependent L-tryptophan oxidase and VioB, a heme protein, work in combination to oxidize and dimerize L-tryptophan to an IPA imine dimer. Hydrogen peroxide is released as a by-product of the VioA reaction. Next step by VioE is the rearrangement of the IPA imine dimer to prodeoxyviolacein acid, which can non-enzymatically oxidise to prodeoxyviolacein or, by VioC via deoxyviolaceinic acid, oxidase to pink deoxyviolacein. The flavin-dependent oxygenases VioC and VioD contain 19 nucleotide binding amino acids, which require interaction with the oxidized form of FAD (flavin-adenine dinucleotide). The two enzymes act sequentially: first, VioD hydroxylates protodeoxyviolaceinic acid, leading to protoviolaceinic acid. Second, VioC creates the oxindole at the 2-position of one indole ring, leading to violet violacein.
Validation
This part is validated through sequencing and test digest. It's functionality is proven in yeast via western blot. Furthermore an in vitro enzyme assay together with VioA and VioB, producing violaceins intermediate prodeoxyviolacein, was performed successfully.
Testdigest
The digest was performed with EcoRI and SacI, fragments are expected at 900 bp and 3115 bp. The replicates Nr. 2 and Nr. 4 are submitted.
Experiments
The functionality of the VioE was proved in yeast via western blot. Furthermore an in vitro enzyme assay together with VioA and VioB, producing violaceins intermediate prodeoxyviolacein, was performed successfully.
Expected size: 22,7 kDa. The Anti-His tag was used for identification.
Prodeoxyviolacein
Prodeoxyviolacein shows a characteristic green color shape. VioE is the last enzyme in the 3-enzyme-pathway of prodeoxyviolacein production.
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