Naur Peter, Hansen Carsten Hørslev, Bak Søren, Hansen Bjarne Gram, Jensen Niels Bjerg, Nielsen Hanne Linde, Halkier Barbara Ann
Plant Biochemistry Laboratory, Department of Plant Biology and Center for Molecular Plant Physiology (PlaCe), The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg, Copenhagen, Denmark.
Arch Biochem Biophys. 2003 Jan 1;409(1):235-41. doi: 10.1016/s0003-9861(02)00567-2.
The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.
来自白芥的细胞色素P450 CYP79B1已在大肠杆菌中实现异源表达，并被证明可催化色氨酸转化为吲哚-3-乙醛肟。构建了三种表达载体，一种表达天然蛋白，另外两种表达具有不同N端修饰的蛋白。以每升培养物的酶活性估算，天然载体的产量最高。将在大肠杆菌中异源表达的拟南芥NADPH：细胞色素P450还原酶ATR1与表达CYP79B1的大肠杆菌原生质球进行重组，以获得酶活性。这表明大肠杆菌的电子供体系统，即黄素氧还蛋白/黄素氧还蛋白还原酶，不支持CYP79B1的活性。重组CYP79B1对色氨酸的K(m)为29±2 microM，V(max)为36.5±0.7nmol h(-1)(ml培养物)(-1)。CYP79B1与拟南芥的CYP79B2和CYP79B3在氨基酸水平上的同一性分别为93%和84%，与甘蓝型油菜的CYP79B5（登录号AF453287）的同一性为96%。细胞色素P450的CYP79B亚家族可能在吲哚硫代葡萄糖苷的生物合成中构成一组直系同源基因。
