Part:BBa_K2043001

catA from Acinetobacter pittii, codon optimized for E. coli

This part corresponds to the gene coding for the protein Catechol-1,2-dioxygenase cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project.
This enzyme is an intradiol dioxygenase that catalyses oxidative ring cleavage of catechol. EC number is 1.13.11.1

Figure 1
Figure 1 shows the action of Catechol-1,2-dioxygenase in degrading the phenolic ring of catechol, resulting in the opening of this molecule. By similarly degrading the phenol rings of anthocyanins, this enzyme should remove reduce anthocyanin pigmentation.

Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds are structurally similar to catechol, making Catechol-1,2-dioxygenase a good candidate for anthocyanin degradation. Catechol-1,2-dioxygenase is also found in many species of soil bacteria. This enzyme originally comes from Acinetobacter pittii (NCBI Ref. Seq.: YP_004995593.1), which we codon optimized for E. Coli and avoided the BsaI restriction sites. An His-tag was also added at the C-terminal. This tag allows for purification in an easier way. We wanted to test Catechol-dioxygenases: one was CatA from Acinetobacter pittii, which uses catechol as a main substrate. We hypothesized that this enzyme would be a strong candidate for removal of red-wine stains because catechol shares important structural similarities with anthocyanin (Cerdan 1995, Kobayasi 1995 and Lin 2015).

Testing the part

We tested the expression and activity of CatA using cell extract of cells expressing our protein.
First, we performed an SDS-PAGE to check whether the protein was being expressed.


Figure 2
Although we did not observe expression of the enzyme (Figure 2), we tested the cell extract for CatA activity in 50 mM sodium phosphate at pH 7 using 30 mM catechol as a substrate, following the protocol from the literature (Lin, 2015) (Figure 3).
The control represents cell extract from E. coli not producing the enzyme. In all cases, values measured correspond to the oxidized catechol reaction product, measured at 260 nm.

Figure 3
Based on this result, we can conclude that our codon-optimized enzymes degrades catechol to its reaction product.

References
Lin, J., & Milase, R. N. (2015). Purification and Characterization of Catechol 1, 2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. The protein journal, 34(6), 421-433.

Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.

NCBI Reference Sequence: YP_004995593.1

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]