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.di-iron enzymes ͉ metalloenzymes ͉ N-oxygenation ͉ reaction mechanism

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…By measuring whole-cell activities of Streptomyces lividans containing genes for wild-type and deletion variants of the aureothin pathway on expression plasmids, AurF was proposed to sequentially oxidize p-aminobenzoate to p-nitrobenzoate (66,206). AurF was independently purified and characterized, showing that p-hydroxylaminobenzoate and p-nitrosobenzoate are intermediates of the reaction (28). Analysis of crystal structures, combined with previous biochemical spectroscopy studies, confirmed that AurF contains two iron atoms in the active site (28,176).…”

“…AurF was independently purified and characterized, showing that p-hydroxylaminobenzoate and p-nitrosobenzoate are intermediates of the reaction (28). Analysis of crystal structures, combined with previous biochemical spectroscopy studies, confirmed that AurF contains two iron atoms in the active site (28,176). The only other characterized homologs of AurF are NorF from Streptomyces orinoci HKI-0260 and SpnF from Streptomyces spectabilis, which are responsible for the biosynthesis of orinocin in S. orinoci (195) and of neoaureothin (spectinabilin) in S. spectabilis (27), respectively.…”

Expression and characterization of an <i>N</i>-oxygenase from <i>Rhodococcus jostii</i> RHAI

“…There have been conflicting reports regarding the structure of AurF, some suggesting the active site contains diiron while others suggesting an iron and manganese complex (Krebs et al, 2007;Zocher et al, 2007). While some structural and biochemical data suggest that AurF may employ a binuclear manganese center, Choi et al (2008) indicated that a diiron center is more likely in the Streptomyces thioluteus AurF. In contrast, PrnD is a Rieske mononuclear non-heme enzyme that catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group, forming pyrrolnitrin.…”

“…An inspection of a protein alignment amongst the three proteins revealed that the characteristic iron binding EX28-33DEX2H motif (Krebs et al, 2007) is present in RHAIro06104. Analysis of the crystal structure of the AurF protein (PDB ID: 3CHH_B) from S. thioluteus (Choi et al, 2008) indicates that the amino acids in this binding motif at positions 200-202 are closely associated with the diiron center. The residues believed to comprise the active site pocket showed amino acid substitutions that were conserved based on similar side chain chemistries (Fig.…”

“…AurF and PrnD were the first examples of N-oxygenases described, each of which are involved in the biosynthesis of aureothin and pyrrolnitrin, respectively (Choi et al, 2008;He et al, 2001;Kirner et al, 1998;Krebs et al, 2007;Lee et al, 2005;Simurdiak et al, 2006;Zhang and Parry, 2007). AurF catalyzes the formation of p-nitrobenzoate from paminobenzoate, an unusual component of the aureothin polyketide synthase.…”