Part:BBa_K2461000

Bromoperoxidase Composite Part (With His6)

This part is a composite part made with the constitutive promoter BBa_j23119, the ribosome binding site BBa_j61100, the gene that codes for a vanadium dependent bromoperoxidase enzyme, and the commonly used terminator BBa_B0015.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 230
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1295
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 452
  Illegal SapI.rc site found at 176
  Illegal SapI.rc site found at 1373
  Illegal SapI.rc site found at 1781
  

Vanadium-Dependent Bromoperoxidase and Our application

Figure 1:Proposed catalytic cycle of the vanadium dependent bromoperoxidase.

Vanadium dependent bromoperoxidase is a kind of haloperoxidase that is involved in the bromination of organic halo-compounds associated with defense and pigmentation in seaweeds and marine algae. The active site features a vanadium oxide center attached to the protein via one histidine side chain and a collection of hydrogen bonds to the oxide ligands. The enzyme can use many different hydrocarbon substrates to catalyse the oxidation of bromide by hydrogen peroxide. The resulting electrophilic bromonium cation (Br+) attacks hydrocarbons (symbolized as R-H in the following equation): R-H + Br+ + H2O2 → R-Br + H2O + OH−

We took this specific bromoperoxidase sequence from the marine algae species Corallina pilulifera. We picked this specific species because it has been proven that feeding Corallina pilulifera to cattle can reduce methane emissions by up to 50%.[1] Each cow releases 70 kg to 120 kg of methane annually, and coupled with the sheer magnitude of the cattle industry this has become a problem of increasing importance. We will use the vanadium-dependent bromoperoxidase enzyme to create bromoform; the main compound in seaweed that is responsible for decreasing methane emissions in cattle. Bromoform works by inhibiting the efficiency of the methyltransferase enzyme by reacting with the reduced vitamin B12 cofactor required for the second to last step of methanogenesis.

[1]. Kinley, R. D., and Fredeen, A. H. (2014) In vitro evaluation of feeding North Atlantic storm toss seaweeds on ruminal digestion. Journal of Applied Phycology 27, 2387–2393.

Figure 2: Absorbance (290) vs. Time (s) of the cell lysate solution composed of monochlorodimedone, potassium bromide, MOPS buffer, and cell lysate containing the vanadium-dependent bromoperoxidase enzyme. The reaction was catalyzed with 20 µL of hydrogen peroxide.

Figure 3:Absorbance (290) vs. Time (s) of the empty vector solution composed of monochlorodimedone, potassium bromide, MOPS buffer, and cell lysate containing the empty vector backbone pSB1C3. The reaction was catalyzed with 20 µL of hydrogen peroxide.

Results

The monochlorodimedone enzyme assay is commonly used to characterize the bromoperoxidase enzyme. It measures the decrease in absorbance at 290 as bromoperoxidase brominates monochlorodimedone into monobromochlorodimedone using bromide ions. This reaction is catalyzed by hydrogen peroxide.

The adjacent results display that our part is working as expected. When hydrogen peroxide is added to the cell lysate containing bromoperoxidase there is a negative slope indicating a decrease in absorbance When hydrogen peroxide is added to the empty vector the slope has a positive value indicating an increase in absorbance. Both cell lysates stick to their regression lines roughly the same amount. The bromoperoxidase cell lysate has a R2 value of 0.693 and the empty vector cell lysate has a R2 value of 0.652. This shows that both reactions followed their trend line to a significant degree. This is good news because it means that our data was not random, instead it was being controlled by the bromination of monochlorodimedone into monobromochlorodimedone.

In this trial the cell lysate was found to be converting 0.00094 units of monochlorodimedone per milliliter of enzyme. The cell lysate with the empty vector was converting -0.0019 units of monochlorodimedone per milliliter of enzyme. This makes sense because the empty vector was not brominating monochlorodimedone.