Reconstitution and characterization of aminopyrrolnitrin oxygenase, a Rieske N-oxygenase that catalyzes unusual arylamine oxidation

Jungkul Lee, Michael Simurdiak, Huimin Zhao

Research output: Contribution to journalArticlepeer-review

Abstract

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, Cys 88, His71, His91, Asp323, His 186, and His191, respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with Km = 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.

Original languageEnglish (US)
Pages (from-to)36719-36728
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number44
DOIs
StatePublished - Nov 4 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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