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PLoS One. 2016 Oct 5;11(10):e0162578. doi: 10.1371/journal.pone.0162578. eCollection 2016.

Mechanism of Rifampicin Inactivation in Nocardia farcinica.

Author information

1
Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, United States of America.
2
Department of Chemistry, Faculty of Science, Damietta University, Damietta, 34517, Egypt.
3
Fralin Life Science Institute, Virginia Tech, Blacksburg, VA, 24061, United States of America.
4
Department of Food Technology, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications, Alexandria, 21934, Egypt.
5
Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA, 24061, United States of America.

Abstract

A novel mechanism of rifampicin (Rif) resistance has recently been reported in Nocardia farcinica. This new mechanism involves the activity of rifampicin monooxygenase (RifMO), a flavin-dependent monooxygenase that catalyzes the hydroxylation of Rif, which is the first step in the degradation pathway. Recombinant RifMO was overexpressed and purified for biochemical analysis. Kinetic characterization revealed that Rif binding is necessary for effective FAD reduction. RifMO exhibits only a 3-fold coenzyme preference for NADPH over NADH. RifMO catalyzes the incorporation of a single oxygen atom forming an unstable intermediate that eventually is converted to 2'-N-hydroxy-4-oxo-Rif. Stable C4a-hydroperoxyflavin was not detected by rapid kinetics methods, which is consistent with only 30% of the activated oxygen leading to product formation. These findings represent the first reported detailed biochemical characterization of a flavin-monooxygenase involved in antibiotic resistance.

PMID:
27706151
PMCID:
PMC5051949
DOI:
10.1371/journal.pone.0162578
[Indexed for MEDLINE]
Free PMC Article

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