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 Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.
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. A 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 SDS-PAGE gels for expression of BpuI, CatA and XylE. Sample preparation: E.coli BL21(DE3) cells expressing the proteins were induced for 5 hours with 0.5mM IPTG. After 5 hours, the OD(600nm) was measured, the cells centrifuged and the pellet was resuspended in Laemmili sample buffer to a final OD of 10. The cells were cooked at 95ºC for 10 min, and 10uL of the resulting solution were loaded on the gel. Ladder used: Kaleidoskope. Staining: The gel was washed 3x with miliQ water to remove the SDS and staining was performed using BioRad Comassie for 30min. De-staining was performed by leaving the gel in miliQ water for 1 hour with gentle shaking. As expected, no overexpression was observed in BL21(DE3), whether it was induced or not induced. BpuI was correctly overexpressed, with the observed size being the expected, around 59kDa. CatA overexpression was very mild, but the correct sized band was observed, of around 34kDa. XylE was correctly overexpressed, with the correct band being observed, of around 36kDa.
The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity. We tested our cell extract for CatA activity in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product.
Figure 3 CatA activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed.
As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product, cis,cis-muconic, at 260nm. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional.
References
Cerdan, P., Rekik, M., & Harayama, S. (1995). Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida. Eur J Biochem, 229(1): 113-8Kobayashi, T., Ishida, T., Hoiike, K., Takahara, Y., Numao, N., Nakazawa, A., Nakazawa, T., & Nozaki, M. (1995). Overexpression of Pseudomonas putida catechol 2,3-dioxygenase with high specific activity by genetically engineered Escherichia coli. J Biochem, 117(3): 614-22
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.
Sequence and Features
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