Regio‐ and Chemoselective Enzymatic N‐Oxygenation In Vivo, In Vitro, and in Flow

Abstract

Action by the para: Evaluation of the nitro‐group‐forming N‐oxygenase AurF in vivo, in vitro, and immobilized as a fusion protein with simply H2O2 as oxidant (peroxide shunt) reveals para‐regioselective oxygenation of aromatic amines (see scheme). This effect includes the selective oxygenation of diamino compounds.

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.…”

“…The other, aminopyrrolnitrin N-oxygenase (PrnD), a Rieske mononuclear non-heme iron enzyme involved in the biosynthesis of pyrrolnitrin, catalyzes the conversion of aminopyrrolnitrin to pyrrolnitrin (3,5). Notably, AurF shares no sequence homology with any other functionally characterized oxygenases in the Swiss-Prot database, and the purified enzyme did not show any native enzymatic activity (6,7), although a ''peroxide shunt'' was recently found to be able to restore the enzymatic activity (8).…”

“…A combination of experimental results from bioinformatics, inductively coupled plasma (ICP) mass spectrometry (MS), and electron paramagnetic resonance (EPR) suggested that AurF is a di-iron enzyme (6). In sharp contrast, Hertweck and coworkers recently concluded that AurF is a di-manganese enzyme on the basis of ICP-MS data of a maltose binding protein-AurF fusion protein (8), crystal structure of AurF in an oxidized state and anomalous x-ray diffraction (9), and structure-based site-directed mutagenesis (7). As an attempt to reconcile the apparently contradictory results between the two studies, Bollinger and coworkers proposed that AurF may contain a heterodinuclear Mn/Fe cofactor (10), similar to the R2 subunit of class Ic ribonucleotide reductase from Chlamydia trachomatis (11).…”

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

“…The K cat value for 4-aminophenol was 0.38 s -1 (Table 2) which was the same that was reported for PsAAO, however RHAI-ro06104 showed activity with fewer substrates than PsAAO or AurF. RHAI-ro06104 did not exhibit strong para regioselectivity compared to AurF (Winkler et al, 2006). Activity was observed with both para and ortho hydroxyl groups (4 and 2-aminophenol, respectively).…”

“…More recently, other putative N-oxygenases have been characterized, FrbG (Johannes et al, 2010), CmlI (Lu et al, 2012;Makris et al, 2010) and PsAAO (Platter et al, 2011). The few enzymes characterized thus far have demonstrated limited substrate specificities with a preference for paminobenzoates and o-aminophenols (Platter et al, 2011;Winkler et al, 2006).…”