title: Mechanism of Rifampicin Inactivation in Nocardia farcinica.
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.
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, United States of America.|Department of Chemistry, Faculty of Science, Damietta University, Damietta, 34517, Egypt.
authors.forename: Heba
authors.initials: H
authors.lastname: Abdelwahab
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, United States of America.|Fralin Life Science Institute, Virginia Tech, Blacksburg, VA, 24061, United States of America.
authors.forename: Julia S
authors.initials: JS
authors.lastname: Martin Del Campo
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, United States of America.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Dai
authors.affiliation: Department of Chemistry, Faculty of Science, Damietta University, Damietta, 34517, Egypt.
authors.forename: Camelia
authors.initials: C
authors.lastname: Adly
authors.affiliation: Department of Food Technology, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications, Alexandria, 21934, Egypt.
authors.forename: Sohby
authors.initials: S
authors.lastname: El-Sohaimy
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, United States of America.|Fralin Life Science Institute, Virginia Tech, Blacksburg, VA, 24061, United States of America.|Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA, 24061, United States of America.
authors.forename: Pablo
authors.initials: P
authors.lastname: Sobrado
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009615:Nocardia / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012293:Rifampin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D010100:Oxygen; D012293:Rifampin