Among the 27 cytochrome P450s (CYPs) of Nocardia farcinica IFM10152, three CYPs have been identified as having O-dealkylation catalytic activity. Of the two that encode CYP154 subfamilies, the one encoded by the nfa22930 gene showed distinct O-dealkylation and subsequent hydroxylation of formononetin. Firstly, formononetin was O-dealkylated into daidzein, which was subsequently mono-hydroxylated at the 3'-position of the B-ring into ortho-dihydroxy-isoflavone. Apparent k(cat)/K-m values of CYP154 for the O-dealkylation of forrnononetin and the hydroxylation of daidzein were 3.57 and 1.84 mu M-1 min(-1), respectively. The dissociation constants of CYP154 based on spectral changes upon binding to each substrate were 5.16 and 3.11 mu M, respectively. Homology modeling and docking simulation found that Thr247 is responsible for the 3'-position hydroxylation reaction by forming a hydrogen bond with the 4'-hydroxyl group of daidzein that forces the proton at the 3'-position to face the heme center. Site-directed mutagenesis of Thr247 to alanine drastically decreased the binding affinity for daidzein (9.73 mu M) as well as 3'-position hydroxylation catalytic activity by 3 fold (0.48 mu M-1 min(-1)). (C) 2010 Elsevier Inc. All rights reserved.