title: Identification of new substrates for the CYP106A1-mediated 11-oxidation and investigation of the reaction mechanism.
abstract: CYP106A1 from Bacillus megaterium DSM319 was recently shown to catalyze steroid and terpene hydroxylations. Besides producing hydroxylated steroid metabolites at positions 6β, 7β, 9α and 15β, the enzyme displayed previously unknown 11-oxidase activity towards 11β-hydroxysteroids. Novel examples for 11-oxidation were identified and confirmed by (1)H and (13)C NMR for prednisolone, dexamethasone and 11β-hydroxyandrostenedione. However, only 11β-hydroxyandrostenedione formed a single 11-keto product. The latter reaction was chosen to investigate the kinetic solvent isotope effect on the steady-state turnover of the CYP106A1-mediated 11-oxidation. Our results reveal a large inverse kinetic isotope effect (∼0.44) suggesting the involvement of the ferric peroxoanion as a reactive intermediate.
authors.affiliation: Institute of Biochemistry, Saarland University, D-66123 Saarbruecken, Germany.
authors.forename: Flora Marta
authors.initials: FM
authors.lastname: Kiss
authors.affiliation: Institute of Biochemistry, Saarland University, D-66123 Saarbruecken, Germany.
authors.forename: Yogan
authors.initials: Y
authors.lastname: Khatri
authors.affiliation: Institute of Pharmaceutical Biology, Saarland University, D-66123 Saarbruecken, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Zapp
authors.affiliation: Institute of Biochemistry, Saarland University, D-66123 Saarbruecken, Germany. Electronic address: ritabern@mx.uni-sarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000735:Androstenedione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001410:Bacillus megaterium / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D012997:Solvents; D000735:Androstenedione; C003582:11-hydroxyandrostenedione; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium
keywords: 11-Oxidation; 11β-Hydroxysteroid; CYP106A1; Cytochrome P450; Ferric peroxoanion; Kinetic solvent isotope effect