Identification of new substrates for the CYP106A1-mediated 11-oxidation and investigation of the reaction mechanism

CYP106A1 from Bacillus megaterium DSM319 was recently shown to catalyze steroid and terpene hydroxylations. Besides producing hydroxylated steroid metabolites at positions 6 beta, 7 beta, 9 alpha and 15 beta, the enzyme displayed previously unknown 11-oxidase activity towards 11 beta-hydroxysteroids. Novel examples for 11-oxidation were identified and confirmed by H-1 and C-13 NMR for prednisolone, dexamethasone and 11 beta-hydroxyandrostenedione. However, only 11 beta-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 (similar to 0.44) suggesting the involvement of the ferric peroxoanion as a reactive intermediate. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.