Part:BBa_K4447003

RifMo coding sequence

Rifampicin monooxygenase coding sequence from Nocardia farcinica. This enzyme catalyzes the oxidation of rifampicin, thereby inactivating its antibiotic activity. It constitutes a secondary rifampicin resistance factor.

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 1225
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1456
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

In our project, rifampicin monooxygenase (EC 1.14.13.211) is used as a detector for the presence of rifampicin by catalyzing the hydroxylation of rifampicin to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. 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 rifampicin through a coupled reaction employing a NADP+/NADPH colorimetric assay.

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.