Mechanism of Rifampicin Inactivation in <i>Nocardia farcinica</i>

<div><p>A novel mechanism of rifampicin (Rif) resistance has recently been reported in <i>Nocardia farcinica</i>. 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.</p></div

Flavin oxidation.

<p>(A) <i>k</i>_obs values as a function of [O₂] without Rif (closed circles) and with 15 μM Rif (opened circles). (B) Spectra changes during the oxidation of free reduced RifMO with 250 μM O₂. The inset shows the changes in absorbance at 450 nm as a function of oxygen concentration. (C) Spectra changes during the oxidation of free, reduced RifMO with 200 μM O₂. The inset shows changes in absorbance at 450 nm as a function of oxygen concentration. Both data were fit to a single phase exponential equation.</p

Time-dependent HPLC analysis of RifMO reactions.

<p>(A) Stacked chromatograms showing time traces for the elution of the Rif peak (21.2 min), P* (13.4 min), Rif-OH (22.1 min), and the P* degradation compound (6.7 min). (B) Stacked chromatograms show P* (A) extracted in 100 mM sodium phosphate buffer, pH 7.5, incubated with: (B) NADPH, (C) RifMO, (D, E) NADPH and RifMO, for 5, and 20 min., respectively.</p

Reaction catalyzed by RifMO.

<p>Reaction catalyzed by RifMO.</p

Activity of RifMO monitored by oxygen consumption and HPLC.

<p>Activity of RifMO monitored by oxygen consumption and HPLC.</p

Individual UV-spectra extracted for pure peaks during HPLC analysis, representing all species involved in the RifMO reaction.

<p>(A) Rif, (B)The first product, (P*), (C) The final product, Rif-OH, and (D) Rif degradation compound of the first product. Rif-OH structure was elucidated from the NMR analysis.</p

TLC monitoring of RifMO activity with Rif.

<p>Lane 1, Rif; lane 2, P*-generating reaction; lane 3, Rif-OH-generating reaction.</p

Spectral changes during Rif binding and turnover.

<p>(A) Flavin spectral changes as a function of increasing concentration of Rif (0–40 μM). The spectral changes show increases in absorbance at (320, 360, 400, 443, and 525 nm) and the isobestic point at ~ 480 nm. The inset shows spectral differences after subtracting the spectrum of RifMO with 0 μM Rif. (B) Determination of the <i>K</i>_D value of Rif. The change in absorbance of the RifMO-Rif complex at 525 nm was plotted as a function of Rif to determine a <i>K</i>_D value. (C) UV-Vis spectrophotometric monitoring of RifMO product formation representing the decline of the Rif peak at 475 nm (blue), followed by red shifting and an absorbance increase at 493 nm (red).</p

Rapid reaction kinetic parameters.

<p>Rapid reaction kinetic parameters.</p

Flavin reduction with NADPH.

<p>Spectra changes for the substrate-free RifMO with 2 mM NADPH. (B) Change in the flavin absorbance at 450 nm for substrate-free RifMO at various concentrations of NADPH (0.025–2 mM). (C) Dependence of the <i>k</i>_obs values as a function of NADPH in the presence of 15 μM Rif. Data was fit to a single exponential decay equation. (D) Spectra changes for the Rif-RifMO complex with 2 mM NADPH. (E) Change in the flavin absorbance at 450 nm for substrate-complexed RifMO at various concentrations of NADPH (0.025–5 mM). (F) Dependence of the <i>k</i>_obs values as a function of NADPH in the absence of Rif. Data was fit to a single exponential decay equation.</p