Abstract
Two-component oxygenases catalyze a wide variety of important oxidation reactions. Recently we characterized a novel arylamine N-oxygenase (PrnD), a new member of the two-component oxygenase family (J. Lee et al., J. Biol. Chem. 280:36719-36728, 2005). Although arylamine N-oxygenases are widespread in nature, aminopyrrolnitrin N-oxygenase (PrnD) represents the only biochemically and mechanistically characterized arylamine N-oxygenase to date. Here we report the use of bioinformatic and biochemical tools to identify and characterize the reductase component (PrnF) involved in the PrnD-catalyzed unusual arylamine oxidation. The prnF gene was identified via sequence analysis of the whole genome of Pseudomonas fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. The purified PrnF protein catalyzes reduction of flavin adenine dinucleotide (FAD) by NADH with a k(cat) of 65 s(-1) (K(m) = 3.2 muM for FAD and 43.1 muM for NADH) and supplies reduced FAD to the PrnD oxygenase component. Unlike other known reductases in two-component oxygenase systems, PrnF strictly requires NADH as an electron donor to reduce FAD and requires unusual protein-protein interaction with the PrnD component for the efficient transfer of reduced FAD. This PrnF enzyme represents the first cloned and characterized flavin reductase component in a novel two-component arylamine oxygenase system.