Identification and characterization of the flavin:NADH reductase (PrnF) involved in a novel two-component arylamine oxygenase.
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.
Lee
Jung-Kul
JK
University of Illinois at Urbana-Champaign, Department of Chemical and Biomolecular Engineering, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Zhao
Huimin
H
D001426:Bacterial Proteins / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D011485:Protein Binding; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics*; D011764:Pyrrolnitrin / Q000096:biosynthesis; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005415:Flavins; D009601:Nitroreductases; D011764:Pyrrolnitrin