title: Biotransformation of isoflavone using enzymatic reactions.
abstract: The roles of cytochrome P450 monooxygenases (CYPs) from Streptomyces spp. which are called the "treasure islands" for natural products for medicine and antibiotics are not well understood. Substrate specificity studies on CYPs may give a solution for elucidation of their roles. Based on homology sequence information, the CYP105D7 of a soluble cytochrome P450 known as heme protein from Streptomyces avermitilis MA4680 was expressed using the T7 promoter of the bacterial expression vector pET24ma, over-expressed in Escherichia coli system and characterized. An engineered whole cell system for daidzein hydroxylation was constructed using an exogenous electron transport system from ferredoxin reductase (PdR) and ferredoxin (Pdx). Also, an in vitro reaction study showed the purified CYP105D7 enzyme, using NADH-dependent-reducing equivalents of a redox partner from Pseudomonas putida, hydroxylated daidzein at the 3' position of the B ring to produce 7,3,'4' trihydroxyisoflavone. The hydroxylated position was confirmed by GC-MS analysis. The turnover number of the enzyme was 0.69 μmol 7,3,'4'-trihydroxyisoflavone produced per μmol P450 per min. This enzyme CYP105D7 represents a novel type of 3'-hydroxylase for daidzein hydroxylation. A P450 inhibitor such as coumarin significantly (ca.98%) inhibited the daidzein hydroxylation activity.
authors: Division of Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong-dong, Jeongeup, Jeonbuk 580-185, Korea. chroh@kaeri.re.kr
authors: Changhyun
authors: C
authors: Roh
mesh_terms: D000595:Amino Acid Sequence; D001711:Biotransformation; D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006900:Hydroxylation / Q000187:drug effects; D007529:Isoflavones / Q000737:chemistry / Q000378:metabolism*; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D016415:Sequence Alignment
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007529:Isoflavones; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D007654:Ketoconazole