Structure and Action of the N-oxygenase AurF from Streptomyces thioluteus

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

“…The protein with a Mn-free di-iron center gave maximal activity and the gradual decrease of iron content in AurF led to the gradual decrease of in vitro enzymatic activity (Table 1). Contrary to the finding by the Hertweck group that ''manganese was enriched by a factor of Ϸ20 in relation to iron'' in purified AurF proteins (8,9), our data clearly show a high selectivity of the protein for iron (rows 3 and 4 in Table 1). This discrepancy is likely due to the use of Fe(III) citrate hydrate and unusually high concentrations of iron and manganese in the culture media in the previous study rather than Fe(NH 4 ) 2 (SO 4 ) 2 , which was used in our experiments and many other similar studies involving di-iron enzymes.…”

“…Additionally, in this earlier report, modeling of substrate pABA into the active site of manganese AurF required significant conformational alterations of the side chains of Arg-96, Leu-202, and Phe-264 (9). Hence, substitution of manganese at the di-metal center would most likely result in an inactive enzyme, consistent with our biochemical observations.…”

“…Enzyme kinetic assays, using reduced AurF prepared by FdR/Fd, were carried out at 20°C in 1 ml of reaction vials of 20 mM Hepes (pH 7.4). The (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.…”

“…During the preparation of this article, the Schulz and coworkers (9) reported that the structure of manganese substituted AurF from protein samples cultivated under high concentrations of this divalent metal. Although this structure unequivocally demonstrates a di-manganese center, there are significant notable differences in the active site architecture compared with our structure of di-iron AurF (Figs.…”

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

“…AurF catalyzes the formation of p-nitrobenzoate from paminobenzoate, an unusual component of the aureothin polyketide synthase. 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.…”