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J. Biol. Chem., Vol. 280, Issue 44, 36719-36727, November 4, 2005

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Reconstitution and Characterization of Aminopyrrolnitrin Oxygenase, a Rieske N-Oxygenase That Catalyzes Unusual Arylamine Oxidation^*

From the Departments of Chemical and Biomolecular Engineering and Chemistry, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801

Rieske oxygenases catalyze a wide variety of important oxidation reactions. Here we report the characterization of a novel Rieske N-oxygenase, aminopyrrolnitrin oxygenase (PrnD) that catalyzes the unusual oxidation of an arylamine to an arylnitro group. PrnD from Pseudomonas fluorescens Pf5 was functionally expressed in Escherichia coli, and the activity of the purified PrnD was reconstituted, which required in vitro assembly of the Rieske iron-sulfur cluster into the protein and the presence of NADPH, FMN, and an E. coli flavin reductase SsuE. Biochemical and bioinformatics studies indicated that the reconstituted PrnD contains a Rieske iron-sulfur cluster and a mononuclear iron center that are formed by residues Cys⁶⁹, Cys⁸⁸, His⁷¹, His⁹¹, Asp³²³, His¹⁸⁶, and His¹⁹¹, respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with K_m = 191 µM and k_cat = 6.8 min^–1. Isotope labeling experiments with ¹⁸O₂ and H₂¹⁸O suggested that the oxygen atoms in the pyrrolnitrin product are derived exclusively from molecular oxygen. In addition, it was found that the oxygenation of the arylamine substrates catalyzed by PrnD occurs at the enzyme active site and does not involve free radical chain reactions. By analogy to known examples of arylamine oxidation, a catalytic mechanism for the bioconversion of amino pyrrolnitrin into pyrrolnitrin was proposed. Our results should facilitate further mechanistic and crystallographic studies of this arylamine oxygenase and may provide a new enzymatic route for the synthesis of aromatic nitro compounds from their corresponding aromatic amines.

Received for publication, May 16, 2005 , and in revised form, August 10, 2005.

^* This work was supported by Office of Naval Research Grant N00014-02-1-0725. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table and supplemental figures.

¹ Supported in part by the Korea Research Foundation Grant funded by Korea Government (Basic Research Promotion Fund Grant MOI-2004-000-10159-0).

² To whom correspondence should be addressed: Dept. of Chemical and Biomolecular Engineering and Chemistry, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801. Tel.: 217-333-2631; Fax: 217-333-5052; E-mail: zhao5{at}uiuc.edu.

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