School of Chemical and Biological Engineering, Institute of Bioengineering, Seoul National University, Seoul, South Korea.
Microb Cell Fact. 2012 Jun 14;11:81. doi: 10.1186/1475-2859-11-81.
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 k(cat)/K(m) 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.
一种细胞色素 P450（CYP）酶，3'-大豆苷元羟化酶，CYP105D7（3'-DH），负责大豆苷元在 3'-位的羟化，最近有报道。CYP105D7（3'-DH）是一种 I 类 CYP，需要通过电子转移蛋白如铁氧还蛋白和铁氧还蛋白还原酶提供电子。目前，我们构建了一种人工 CYP，以开发用于生产羟化产物的反应宿主。通过与自给自足的 CYP102D1 的还原酶结构域进行融合介导构建，以提高电子转移效率并与氧化过程偶联。人工自给自足的大豆苷元羟化酶（3'-ASDH）表现出黄素蛋白和 CYP 的明显光谱特性。融合酶更有效地催化大豆苷元的羟化，k(cat)/K(m) 值为 16.8 μM(-1) min(-1)，约比 3'-DH-camA/B 重建酶高 24 倍。最后，开发了一种用于表达 3'-ASDH 和生产羟化产物的重组链霉菌avermitilis 宿主。达到的转化率（34.6%）比野生型高 5.2 倍。
