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Reconstitution and Characterization of Aminopyrrolnitrin Oxygenase, a Rieske N-Oxygenase That Catalyzes Unusual Arylamine Oxidation
J. Biol. Chem., 280 (44): 36719--36727 (ноября 2005)
DOI: 10.1074/jbc.M505334200
Аннотация
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 Km = 191 microM and kcat = 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. 10.1074/jbc.M505334200
