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

Jungkul Lee,Michael Simurdiak,Huimin Zhao

doi:10.1074/jbc.m505334200

Jungkul Lee, Michael Simurdiak + Show 1 more

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https://doi.org/10.1074/jbc.m505334200

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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 Cys(69), Cys(88), His(71), His(91), Asp(323), His(186), and His(191), respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with K(m) = 191 microM and k(cat) = 6.8 min(-1). Isotope labeling experiments with (18)O(2) and H(2)(18)O 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.

Highlights

The cloning and characterization of a 5.8-kb DNA region that encodes the pyrrolnitrin biosynthetic pathway was reported [8]. This DNA region confers the ability to produce pyrrolnitrin when expressed heterologously in Escherichia coli and contains four genes, prnABCD, each of which is required for pyrrolnitrin production [9]
Arylamine oxygenases seem to be widely distributed within the microbial world and used in a variety of metabolic reactions [11,12,13,14,15,16], PrnD represents one of only two known examples of arylamine oxygenases or N-oxygenases involved in arylnitro group formation, the other being AurF involved in aureothin biosynthesis [16, 17]
To verify the identity of this compound produced by PrnD, it was purified by HPLC as mentioned under “Experimental Procedures,” and this compound was unambiguously identified as pyrrolnitrin by 1H NMR and high resolution electron ionization mass spectrometry. (1H NMR (Acetone-d6, 500 MHz) ␦ 6.91 (m, 1H), 7.02 (m, 1H), 7.65 (m, 4H); HR EI-MS, calculated for pyrrolnitrin (Mϩ) 255.9806, found 255.9808.) 1H NMR spectrum of the isolated product was identical to spectra previously reported for pyrrolnitrin [43]. These results clearly indicate that the product of the prnD gene is an arylamine oxygenase, aminopyrrolnitrin oxygenase

Summary

Introduction

The cloning and characterization of a 5.8-kb DNA region that encodes the pyrrolnitrin biosynthetic pathway was reported [8]. A hypothetical biochemical pathway for the synthesis of pyrrolnitrin has been proposed by van Pee et al [10] In this pathway shown, the first step is the chlorination of tryptophan by PrnA at the 7 position to form 7-chlorotryptophan, followed by rearrangement of the indole ring to a phenylpyrrole ring and decarboxylation by PrnB to form monodechloroaminopyrrolnitrin. The first step is the chlorination of tryptophan by PrnA at the 7 position to form 7-chlorotryptophan, followed by rearrangement of the indole ring to a phenylpyrrole ring and decarboxylation by PrnB to form monodechloroaminopyrrolnitrin This intermediate is chlorinated a second time by PrnC to form aminopyrrolnitrin, which, in the last step of the pathway, undergoes oxidation of the amino group in aminopyrrolnitrin to a nitro group by PrnD to form pyrrolnitrin. We report for the first time the functional expression, purification, reconstitution, and characterization of a novel Rieske N-oxygenase, PrnD, that catalyzes unusual arylamine oxidation.

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