Difference between revisions of "Part:BBa K4447003"
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In our project, rifampicin monooxygenase <b>(EC 1.14.13.211)</b> 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 <b>Figure 1</b>, 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. | In our project, rifampicin monooxygenase <b>(EC 1.14.13.211)</b> 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 <b>Figure 1</b>, 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. | ||
| − | [[Image:RifMo_reaction_TecMonterreyGDL.jpeg| | + | [[Image:RifMo_reaction_TecMonterreyGDL.jpeg|650px|center|thumb|<b>Figure 1</b>. <i>Chemical reaction for rifampicin monooxygenase (RifMo).</i>]] |
Rifampicin monooxygenase, as pictured below in <b>Figure 2</b> is a dimeric protein that has 474 amino acids in length and 51.4 kDa in weight (Hoshino et al., 2010). Koteva and collaborators (2018) reported a Michaelis constant of 12 µM for rifampicin, concluding it has a unique affinity for this substrate. | Rifampicin monooxygenase, as pictured below in <b>Figure 2</b> is a dimeric protein that has 474 amino acids in length and 51.4 kDa in weight (Hoshino et al., 2010). Koteva and collaborators (2018) reported a Michaelis constant of 12 µM for rifampicin, concluding it has a unique affinity for this substrate. | ||
| − | [[Image:RifMo_rotating_TecMonterreyGDL.gif|230px|center|thumb|<b>Figure 2</b>. Three-dimensional structure of RifMo.]] | + | [[Image:RifMo_rotating_TecMonterreyGDL.gif|230px|center|thumb|<b>Figure 2</b>. <i>Three-dimensional structure of RifMo.</i>]] |
=References= | =References= | ||
Latest revision as of 21:43, 9 October 2022
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1225
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1456
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contents
Usage and Biology
Rifampicin is a potent antibiotic against tuberculosis and other mycobacterial infections, but an extensive usage of it and its derivatives has contributed to bacterial resistance, which neutralizes antibiotic activity. The presence of rifampicin in water bodies represents a persintent thread, because of the hazardous potential it has to aquatic organisms and human health (Cai et al., 2019).
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.
Rifampicin monooxygenase, as pictured below in Figure 2 is a dimeric protein that has 474 amino acids in length and 51.4 kDa in weight (Hoshino et al., 2010). Koteva and collaborators (2018) reported a Michaelis constant of 12 µM for rifampicin, concluding it has a unique affinity for this substrate.
References
[1]. Cai, W., Weng, X., & Chen, Z. (2019). Highly efficient removal of antibiotic rifampicin from aqueous solution using green synthesis of recyclable nano-Fe3O4. Environmental pollution (Barking, Essex : 1987), 247, 839–846. https://doi.org/10.1016/j.envpol.2019.01.108
[2]. Hoshino, Y., Fujii, S., Shinonaga, H., Arai, K., Saito, F., Fukai, T., Satoh, H., Miyazaki, Y., & Ishikawa, J. (2010). Monooxygenation of rifampicin catalyzed by the rox gene product of Nocardia farcinica: structure elucidation, gene identification and role in drug resistance. The Journal of antibiotics, 63(1), 23–28. https://doi.org/10.1038/ja.2009.116
[3]. Koteva, K., Cox, G., Kelso, J. K., Surette, M. D., Zubyk, H. L., Ejim, L., Stogios, P., Savchenko, A., Sørensen, D., & Wright, G. D. (2018). Rox, a Rifamycin Resistance Enzyme with an Unprecedented Mechanism of Action. Cell Chemical Biology, 25(4), 403-412.e5. https://doi.org/10.1016/j.chembiol.2018.01.009