In Vitro Reconstitution Reveals a Central Role for the N-Oxygenase PvfB in (Dihydro)pyrazine-N-oxide and Valdiazen Biosynthesis

The Pseudomonas virulence factor (pvf) operon is essential for the biosynthesis of two very different natural product scaffolds: the (dihydro)pyrazine-N-oxides and the diazeniumdiolate, valdiazen. PvfB is a member of the non-heme diiron N-oxygenase enzyme family that commonly convert anilines to their nitroaromatic counterparts. In contrast, we show that PvfB catalyzes N-oxygenation of the α-amine of valine, first to the hydroxylamine and then the nitroso, while linked to the carrier protein of PvfC. PvfB modification of PvfC-tethered valine was observed directly by protein NMR spectroscopy, establishing the intermediacy of the hydroxylamine. This work reveals a central role for PvfB in the biosynthesis of (dihydro)pyrazine-N-oxides and valdiazen.