Part:BBa_K2043006
xylE-FBD10 from Pseudomonas putida codon optimized for E. coli
This part corresponds to Catechol-2,3-dioxygenase (xylE) fused to the Fabric Binding Domain 10 (BBa_K2043017) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzyme catalyses the following chemical reaction with EC number 1.13.11.2
Figure 1 Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.
The figure 1 shows of catechol 2,3-dioxygenases catalyzes the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds have structurally similarties to catechol, specially to the phenolic cycle of anthocyanins, making Catechol-2,3-dioxygenase a good candidate for anthocyanin degradation.
Catechol-2,3-dioxygenase is also found in many species of soil bacteria. This enzymes originally comes from Pseudomonas putida (NCBI Ref. Seq.: NP_542866.1), 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 XylE from Pseudomonas putida , 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 and we expected that the phenolic cycle of the anthocyanin could be a possible target for this enzyme. (Cerdan 1995, and Kobayasi 1995).
The Fabric Binding Domain 10 (FBD10) has affinity for Cellulose. It is positively charged (+1)
Testing the part
We tested our cell extract for XylE as for XylE-FBD10 activity 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 in order to follow the activity of the reaction of XylE and XylE-FB10. The blue line represents the negative control, green line shows the activity of the cell extract containing XylE, and the red line corresponds with the cell extract of cells expressing XylE-FBD10.
Figure 3 XylE fusion proteins' activity. 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 our native and the control, and also between the fusion protein and the control. According with these results we can conclude that the enzyme has activity, however it has been reduced sustanctialy respect to the wild type.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 703
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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