초록
<P>The roles of cytochrome P450 monooxygenases (CYPs) from <I>Streptomyces</I> 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 <I>Streptomyces avermitilis</I> MA4680 was expressed using the T7 promoter of the bacterial expression vector pET24ma, over-expressed in <I>Escherichia coli</I> 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 <I>in vitro</I> reaction study showed the purified CYP105D7 enzyme, using NADH-dependent-reducing equivalents of a redox partner from <I>Pseudomonas putida</I>, 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.</P>