Part:BBa_K4447002

Pcp coding sequence

Pentachlorophenol 4-monooxygenase coding sequence from Flavobacterium sp. This enzyme, a FAD binding and NADPH requiring oxygenase, catalyzes the oxygenolytic removal of the first chlorine from pentachlorophenol.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1651
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Of all the emerging contaminants currently analyzed, pentachlorophenol is one of the most hazardous because of its consequences on human health. Pentachlorophenol has been used as a herbicide, fungicide, disinfectant, and as an ingredient in antifouling paint. Short-term exposure to large doses of pentachlorophenol can cause harmful effects on the liver, blood, lungs, immune system, and gastrointestinal tract (Cai & Xun, 2002).

In our project, pentachlorophenol 4-monooxygenase (EC.1.14.13.50) is used as a detector for the presence of pentachlorophenol by catalyzing the dechlorination of pentachlorophenol to tetrachlorobenzoquinone, requiring NADPH as a reagent and, therefore, obtaining NADP+ as a reaction product. Consequently, it is possible to evaluate the presence of pentachlorophenol through a coupled reaction employing a NADP+/NADPH colorimetric assay. The following image shows the complete reaction according to Hlouchova et al. (2012):

Pentachlorophenol 4-monooxygenase (Pcp) is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. It has 539 amino acids in length and 60.1 kDa in weight (Cai & Xun, 2002). Hlouchova et al. (2012) reported a Michaelis constant of 1 mM for pentachlorophenol, concluding that this enzyme is not well evolved for turnover of this substrate. Nevertheless, this value is smaller than the one for 2,3,5,6-tetrachlorophenol, showing more preference for our desired substrate. Next, we present the three-dimensional structure of Pcp generated by AlphaFold2 using MMSeqs2 (Mirdita et al., 2022). This structure is as follows:

References

[1]. Cai, M., & Xun, L. (2002). Organization and regulation of pentachlorophenol-degrading genes in Sphingobium chlorophenolicum ATCC 39723. Journal of bacteriology, 184(17), 4672–4680. https://doi.org/10.1128/JB.184.17.4672-4680.2002

[2]. Hlouchova, K., Rudolph, J., Pietari, J. M., Behlen, L. S., & Copley, S. D. (2012). Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum. Biochemistry, 51(18), 3848–3860. https://doi.org/10.1021/bi300261p

[3]. Mirdita, M., Schütze, K., Moriwaki, Y. et al.(2022). ColabFold: making protein folding accessible to all. Nat Methods 19, 679–682. https://doi.org/10.1038/s41592-022-01488-1