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Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450cin, CYP176A1) purified from a strain ofCitrobacter braakiithat is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450cingene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450cinmay be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coliat ∼2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coliflavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450cin, it was expressed inE. coliin the presence of cineole 1. A product was formedin vivothat was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450cinby UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (KD= 0.7 μm), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450cincatalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.