The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase

Abstract

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 M. Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed. Rifampicin (RIF)3 ( Fig. 1) is a potent frontline antibiotic against tuberculosis and other mycobacterial infections, but extensive usage of RIF and its derivatives has contributed to bacterial resistance, which neutralizes antibiotic activity (1, 2). In addition to the point mutations in RNA polymerase that are responsible for resistance in mycobacteria (3, 4), some bacterial species, such as soil actinomycetes and parasitic bacteria, employ secondary enzyme-mediated inactivation mechanisms that chemically modify RIF to less active forms or degradation products (5).At least four RIF-deactivating enzymes have been described: ADP-ribosyltransferase (Arr) (6), glycosyltransferase (Rgt) (7,8), phosphotransferase (Rph) (8 -11), and RIF monooxygenase (12, 13). Arr and Rgt act on a critical hydroxyl group (C23) located on the ansa aliphatic chain of RIF, whereas Rph adds a phosphate group to the C21 hydroxyl. These hydroxyls are important for antibiotic action because they hydrogen-bond to a conserved region of the ␤-subunit in RNA polymerase, which is the target of RIF (14). Covalent modification of RIF hydroxyls with ADP-ribose or phosphate results in high level resistance in Escherichia coli, such as a 64-fold increase in the RIF minimal inhibition concentration (6, 10). Modificat...

Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes

“…4d) from the C4a of the flavin cofactor. These distances are similar to C11a of chlortetracycline and C4a of FAD in the Tet(X)+chlortetracycline structure 27 , and well within the C4a-reactive atom distances observed for flavin monooxygenases 39 . The C11a in the Tet(50)+chlortetracycline, on the other hand, is on the opposite side of the molecule and 7.9 Å away (Fig.…”

Rox, a Rifamycin Resistance Enzyme with an Unprecedented Mechanism of Action

“…The 3D Structure of Rox-sv Reveals the FAD Co-factor and Homology to Rox-nf The crystal structures of Rox-sv bound to flavin adenine dinucleotide (FAD; refinement statistics can be found in Table S2) and Rox-sv in complex with both rifampin and FAD were determined to explore active-site similarities between the two homologs (Rox-sv and Rox-nf), which exhibit 74% amino acid sequence identity. The structure of Rox-sv was solved by molecular replacement using Rox-nf (Liu et al, 2016) as a search model. For both the FAD-and rifampin,FAD complexes, the protein crystallized with three molecules in the asymmetric unit; all chains adopted overall similar arrangements (all pairwise rootmean-square deviations [RMSD] less than 0.45 Å ).…”

“…The latter have been detected in a variety of bacteria (Nocardia farcinica, Rhodococcus equi) and are associated with decomposition of the antibiotic. The 3D structure of the N. farcinica rifampin monooxygenase (Rox-nf) was recently reported (Liu et al, 2016). A molecular mechanism for rifampin oxidation was proposed (Abdelwahab et al, 2016;Liu et al, 2016) based on the structure of the isolated product (designated rifampin-2 0 -N-oxide) (Hoshino et al, 2010) ( Figure 1A).…”

“…The 3D structure of the N. farcinica rifampin monooxygenase (Rox-nf) was recently reported (Liu et al, 2016). A molecular mechanism for rifampin oxidation was proposed (Abdelwahab et al, 2016;Liu et al, 2016) based on the structure of the isolated product (designated rifampin-2 0 -N-oxide) (Hoshino et al, 2010) ( Figure 1A). Here we present the identification of a new rifamycin-inducible rox gene in Streptomyces venezuelae (Rox-sv), determination of its 3D structure by X-ray crystallography, and revision of the molecular mechanism of rifamycin inactivation by Rox enzymes.…”

“…Resistance to rifamycin antibiotics in the clinic is predominantly due to point mutations in the target, the b subunit of RNA polymerase (Ramaswamy and Musser, 1998), although a rifampin ADP-ribosyltransferase (Baysarowich et al, 2008) (Arr-2) is found in many Gram-negative resistance plasmids. In contrast to the clinic, environmental bacteria have developed a rich diversity of mechanisms to inactivate the rifamycins including ADP-ribosyltransferases (Baysarowich et al, 2008), glycosyltransferases (Spanogiannopoulos et al, 2012), phosphotransferases (Qi et al, 2016;Spanogiannopoulos et al, 2012;Stogios et al, 2016), and monooxygenases (Andersen et al, 1997;Hoshino et al, 2010;Liu et al, 2016). The latter have been detected in a variety of bacteria (Nocardia farcinica, Rhodococcus equi) and are associated with decomposition of the antibiotic.…”