Part:BBa_K2091000

Protocatechuate 3,4-dioxygenase Alpha Subunit

pcaG is a gene encoding for the Alpha subunit of the Protocatechuate 3,4-dioxygenase enzyme from Pseudomonas putida.

Usage and Biology

Protocatechuate 3,4-dioxygenase catalyzes the convertion of 3,4-dihydroxybenzoic acid and O2 to 3-carboxy-cis,cis-muconate. Dioxygenases are oxydoreductase enzymes. They utilize a dioxygen (O2) molecule as an oxidant. As opposed to monooxygenases, in dioxygenases reactions both atoms from O2 are incorporated into the substrate. Protocatechuate dioxygenase from P. putida has a molecular weight of 200,000 daltons and contains 4 alpha subunits of 23,000 daltons, 4 beta subunits of 26,500 daltons, and 4 ferric irons, suggesting that the enzyme is a tetramer of (𝛂𝛃Fe3+) catalytic units.

Figure 1: The reaction catalyzed by the Protocatechuate 3,4-dioxygenase.

Characterization

The ability of P. putida to utilize protocatechuate (3,4-dihydroxybenzoate) was tested using an assay of M9 minimal medium with varying concentrations of protocatechuate as a sole carbon source (figure 2,3).

Figure 2: Protocatechuate degradation assays. 3,4-dihydroxybenzoate was used as a sole carbon source for the growth of P. putida in a M9 minimal medium in increasing concentrations for a period of 80 hours.

Figure 3: Protocatechuate degradation assays. 3,4-dihydroxybenzoate was used as a sole carbon source for the growth of P. putida in a M9 minimal medium with optimal concentrations based on the previous experiment. Every concentration was tested twice.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 40
  Illegal NgoMIV site found at 79
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 583