Part:BBa_K2043005
xylE-FBD1 from Pseudomonas putida codon optimized for E. coli
This part corresponds to Catechol-2,3-dioxygenase (xylE), EC number 1.13.11.2, fused to Fabric Binding Domain 1 (BBa_K2043010) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzyme catalyses the following chemical reaction:
Figure 1 Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.
Figure 1 shows catechol 2,3-dioxygenases catalyzing the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds have structural similarities to catechol, specially the possession of a phenolic ring.
Catechol-2,3-dioxygenase is also found in many species of soil bacteria. We took this enzyme from Pseudomonas putida (NCBI Ref. Seq.: NP_542866.1), codon optimized the gene for E. coli while removing the BsaI restriction sites. A His-tag was also added at the C-terminal for protein purification.
XylE was one of two Catechol-dioxygenases we tested, in addition to CatA (BBa_K2043001). 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).
The Fabric Binding Domain 1 (FBD1) has affinity for cotton, linen, polyester, silk and wool. It is positively charged (+1) and it is proline rich.
Testing the part
We tested our cell extract for the activity of XylE-FBD1 in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Control corresponds to cells which do not express our proteins. In all cases, values measured correspond to reaction product, 2-hydroxymuconate semialdehyde.
Figure 2 Absorbance of the reaction product, 2-hydroxymunonic semialdehyde. The absorbance of the product was measured at 375nm over a period of time to follow the reaction activity of XylE and XylE-FB1. The blue line represents the negative control, the green line shows the activity of the cell extract containing XylE, and the red line corresponds with the cell extract expressing XylE-FBD.
Figure 3 Measurements were taken after 12 min, timepoint after which all the substrate had been consumed.
As the image indicates, there is a clear difference between the native protein and the control, but no difference between the fusion protein and the control. Based on this, we can conclude that the enzyme lost activity as a result of the fused peptide, or that the addition of this peptide in N-terminal reduces the expression and therefore no XylE-FBD was present in the cell extract.
References
Boyer, S., Biswas, D., Soshee, A. K., Scaramozzino, N., Nizak, C., & Rivoire, O. (2016). Hierarchy and extremes in selections from pools of randomized proteins. Proceedings of the National Academy of Sciences, 201517813.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. European journal of biochemistry, 229(1), 113-118.
Jain, P., Soshee, A., Narayanan, S. S., Sharma, J., Girard, C., Dujardin, E., & Nizak, C. (2014). Selection of arginine-rich anti-gold antibodies engineered for plasmonic colloid self-assembly. The Journal of Physical Chemistry C, 118(26), 14502-14510.
Kobayashi, T., Ishida, T., Horiike, K., Takahara, Y., Numao, N., Nakazawa, A., ... & Nozaki, M. (1995). Overexpression of Pseudomonas putida catechol 2, 3-dioxygenase with high specific activity by genetically engineered Escherichia coli. Journal of biochemistry, 117(3), 614-622.
Soshee, A., Zürcher, S., Spencer, N. D., Halperin, A., & Nizak, C. (2013). General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires. Biomacromolecules, 15(1), 113-121.
Sequence and Features
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- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 700
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