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

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. As shown in Figure 1, this reaction requires NADPH as a reagent and, therefore, gives 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.

Figure 1. Chemical reaction for pentachlorophenol 4-monooxygenase (Pcp).

Pentachlorophenol 4-monooxygenase, as pictured below in Figure 2 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.

Figure 2. Three-dimensional structure of Pcp.

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