In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis

Abstract

p-Aminobenzoate N-oxygenase (AurF) from Streptomyces thioluteus catalyzes the formation of unusual polyketide synthase starter unit p-nitrobenzoic acid (pNBA) from p-aminobenzoic acid (pABA) in the biosynthesis of antibiotic aureothin. AurF is a metalloenzyme, but its native enzymatic activity has not been demonstrated in vitro, and its catalytic mechanism is unclear. In addition, the nature of the cofactor remains a controversy. Here, we report the in vitro reconstitution of the AurF enzyme activity, the crystal structure of AurF in the oxidized state, and the cocrystal structure of AurF with its product pNBA. Our combined biochemical and structural analysis unequivocally indicates that AurF is a non-heme di-iron monooxygenase that catalyzes sequential oxidation of aminoarenes to nitroarenes via hydroxylamine and nitroso intermediates.

Fig. 1.

X-band EPR spectra of the fully reduced AurF at 4 K, using microwave B₁ parallel to the static field B.

Fig. 2.

Structural analysis of AurF. (a) Overall view of the di-iron AurF homodimer shown in a ribbon representation with a color ramp of blue to red (amino to carboxyl terminus). The iron atoms are shown as red spheres. (b) A diagram representation of the superposition of di-iron AurF (shown in blue tube) and di-manganese AurF (shown in pink tube) near the active site. The iron atoms are shown as red spheres, the bridging μ-oxo atom is shown as a green sphere, and the manganese atoms are shown in yellow. (c) Stereoview of the active site of unliganded AurF illustrating the protein ligands to the metal. The iron atoms are shown as dark-red spheres, and the bridging μ-oxo species is shown as a green sphere. Two difference Fourier electron density maps calculated with coefficients |F_obs| − |F_calc| are superimposed on the coordinates. The first map is calculated with phases from the refined model of unliganded AurF minus the two iron atoms and the μ-oxo bridge and is contoured at 3 (colored in magenta) and 11 (colored in yellow) standard deviations above background (σ). The second difference Fourier map was calculated in a similar fashion, but only the bridging μ-oxo atom was omitted before calculations and this map is contoured at 4σ (blue).

Fig. 3.

Stereoview of anomalous difference Fourier electron density maps calculated with phases from the final refined model of AurF minus the two metal atoms and coefficients |F⁺| − |F⁻|, corresponding to wavelength dependent Bijvoet differences. Maps calculated from data collected at the iron absorption edge (λ = 1.7220 Å) are shown in blue (6σ) and magenta (11σ), and maps calculated from data collected at the absorption edge for manganese (λ = 1.8785 Å) are shown in yellow (4σ). The final refined coordinates are superimposed on the maps. The two iron atoms are shown as dark-red spheres, and the bridging μ-oxo atom is shown as a green sphere.

Fig. 4.

Stereoview of difference Fourier electron density maps calculated with coefficients |F_obs| − |F_calc| and phases from the refined model of the AurF-product complex minus the atoms of the partially occupied molecule of pNBA, contoured at 2.3σ over background. The final refined coordinates of the complex are superimposed. The two iron atoms are shown as dark-red spheres, the bridging μ-oxo atom is shown as a green sphere, and the atoms of the ligand are shown in yellow.

Fig. 5.

Structural comparisons. (a) Comparison of the crystal structures of di-iron AurF (in blue) and di-manganese AurF (in pink) (9). The positions of protein residues Arg-96, Ile-199, Leu-202, Leu-203, and Phe-264 vary considerably between the two structures, and these residues are compressed into the active site in the structure of di-manganese AurF. (b and c) The active site of the di-iron AurF contains a well formed cavity that allows for the binding of subtrate without significant changes in the protein scaffold, whereas the active site of di-manganese AurF contains a much smaller cavity and would require rearrangement of residues to accommodate the substrate.

Fig. 6.

HPLC analysis of products in the reaction of 0.1 mM AurF, 0.1 mM PMS, 0.2 mM NADH, and 0.5 mM pABA. Products were identified by authentic sample and LC-MS/MS analysis, and the fragmentation patterns are indicated.