Part:BBa_K3843000

PEA-binding salmon trypsin (1UTM)

Trypsin is a ubiquitous protease in vertebrates due to its central role in digestion. Trypsin cleaves peptide bonds occurring after positively charged amino acids, such as lysine and arginine. 1UTM (pictured below) is a trypsin from Salmo salar (Atlantic salmon). It has a variety of known inhibitors, typically biogenic amines (Leiros et al., 2009). These noncompetitive inhibitors bind to allosteric sites, thereby inactivating trypsin's proteolytic function. Of particular interest to Waterloo iGEM 2021 was the ability of phenylethylamine (PEA, pictured below in green), a dopamine precursor, to act as an inhibitor of 1UTM, with a dissociation constant Kd = 0.000972 (Leiros et al., 2009; RCSB PDB, n.d.). This would allow for 1UTM to be used as a PEA-binding protein. Detection and quantification of phenylethylamine, using 1UTM fused to horseradish peroxidase in a microfluidic flow assay, would give insight into an individual's probability of having ADHD.

3D structure of 1UTM, as per the crystal structure of 1UTM on the PDB (Leiros et al., 2009). 1UTM is coloured in orange, while phenylethylamine (PEA) is coloured in green. The 3D structure was developed using UCSF Chimera (Pettersen et al., 2004).

References

Leiros, H. K. S., Brandsdal, B. O., Andersen, O. A., Os, V., Leiros, I., Helland, R., Otlewski, J., Willassen, N. P., & Smalås, A. O. (2009, January 1). Trypsin specificity as elucidated by lie calculations, x‐ray structures, and association constant measurements. Protein Science, 13(4), 1056-1070. https://onlinelibrary.wiley.com/doi/full/10.1110/ps.03498604.

Male, R., Lorens, J. B., Smalås, A. O., & Torrissen, K. R. (2005, March 3). Molecular cloning and characterization of anionic and cationic variants of trypsin from Atlantic Salmon. European Journal of Biochemistry, 232(2), 677-685. https://febs.onlinelibrary.wiley.com/doi/full/10.1111/j.1432-1033.1995.677zz.x?sid=nlm%3Apubmed.

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C. & Ferrin, T. E. UCSF Chimera--a visualization system for exploratory research and analysis (Version 1.15). J Comput Chem. 2004; 25(13): 1605-1612. https://www.ncbi.nlm.nih.gov/pubmed/15264254

RCSB PDB. (n.d.). 1UTM. Retrieved October 17, 2021, from https://www.rcsb.org/structure/1UTM

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]