Engineering of daidzein 3′-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450.pdfVIP

Choi et al. Microbial Cell Factories 2012, 11:81 /content/11/1/81 RESEARCH Open Access Engineering of daidzein 3’-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450 1 1 1 1 1 2 Kwon-Young Choi , EunOk Jung , Da-Hye Jung , Byeo-Ri An , Bishnu Prasad Pandey , Hyungdon Yun , Changmin Sung3, Hyung-Yeon Park4* and Byung-Gee Kim1* Abstract A cytochrome P450 (CYP) enzyme, 3’-daidzein hydroxylase, CYP105D7 (3’-DH), responsible for daidzein hydroxylation at the 3’-position, was recently reported. CYP105D7 (3’-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3’-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3’-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type. Keywords: Cytochrome P450 monooxygenases, Daidzein 3’-hydroxylase, Self-sufficient P450, CYP102D1 Introduction Fundamentally, t