title: Identification of CYP106A2 as a regioselective allylic bacterial diterpene hydroxylase.
abstract: The cytochrome P450 monooxygenase CYP106A2 from Bacillus megaterium ATCC 13368 catalyzes hydroxylations of a variety of 3-oxo-Δ(4) -steroids such as progesterone and deoxycorticosterone (DOC), mainly in the 15β-position. We combined a high-throughput screening and a rational approach for identifying new substrates of CYP106A2. The diterpene resin acid abietic acid was found to be a substrate and was docked into the active site of a CYP106A2 homology model to provide further inside into the structural basis of the regioselectivity of hydroxylation. The products of the hydroxylation reaction were analyzed by HPLC and the V(max)  and K(m)  values were calculated. The corresponding reaction products were analyzed by NMR spectroscopy and identified as 12α- and 12β-hydroxyabietic acid. CYP106A2 was therefore identified as the first reported bacterial cytochrome P450 diterpene hydroxylase. Furthermore, an effective whole-cell catalyst for the selective allylic 12α- and 12β-hydroxylation was applied to produce the hydroxylated products.
authors.affiliation: Universität des Saarlandes, Biochemie, Campus B2.2, 66123 Saarbrücken, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Bleif
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.forename: Michael
authors.initials: M
authors.lastname: Lisurek
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: von Kries
authors.forename: Josef
authors.initials: J
authors.lastname: Zapp
authors.forename: Matthias
authors.initials: M
authors.lastname: Dietzen
authors.forename: Iris
authors.initials: I
authors.lastname: Antes
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D045784:Abietanes / Q000737:chemistry*; D001410:Bacillus megaterium / Q000201:enzymology*; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry*; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure
chemical_list: D045784:Abietanes; D004224:Diterpenes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C023710:abietic acid