Part:BBa_K2271014

VioA

The synthesis of Violacein requires five enzymes encoded by the VioABCDE operon. VioA is the first enzyme in the violacein pathway. The flavin-dependent L-tryptophan oxidase catalyzes L-tryptophan + O₂ to 2-iminoindole pyruvate + H₂O₂. For this reaction it requires FAD and Mg²⁺ as cofactors. Violacein is naturally produced in gram-negative bacteria strains like Chrombacterium violaceum. In the course of our project we did also work with VioE which is available as a biobrick as well (Part:BBa_K2271017).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1979
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal suffix found in sequence at 2222
  Illegal BglII site found at 860
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

This part is validated via Sequencing, test digest and westernblot. Furthermore an in vitro enzyme assay together with VioB and VioE, producing violaceins intermediate prodeoxyviolacein, was performed successfully. The following figure shows the positive western blot of VioA done with Anti-His Antibody. Expected size: 47.7 kDa

Usage and Biology

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.

Prodeoxyviolacein

Prodeoxyviolacein shows a characteristic green color shape. VioE is the last enzyme in the 3-enzyme-pathway of prodeoxyviolacein production.