Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli
Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli
复制标题
大肠杆菌中表达的细胞色素 P450 物种对小分子的生物转化
DOI：
10.1042/ba20070173
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发表时间：
2008
影响因子：
2.8
通讯作者：
H. Imaishi
中科院分区：
文献类型：
--
作者：
T. Uno;Sota Okamoto;Satoko Masuda;A. Itoh;Y. Uno;Masahiko Nakamura;K. Kanamaru;H. Yamagata;H. Imaishi
P450（细胞色素P450）酶催化多种化合物的单氧作用，如类固醇、脂肪酸、维生素和药物。在本文中，我们展示了一个利用大肠杆菌中表达的P450物种进行多种化合物[黄酮、脱氢表雄酮和7‐乙氧基香豆素]生物转化的系统。首先，我们表达了四个P450物种：兔CYP2B （P450家族2，亚家族B）、果蝇CYP317A、大鼠CYP3A23和小鼠CYP2J5。接下来，我们将底物直接添加到培养液中。用高效液相色谱法和荧光光谱法对所得代谢物进行提取和分析。第一个底物7 -乙氧基香豆素被CYP2B去乙基化；CYP2J5和CYP3A23对7‐乙氧基香豆素的活性较弱，CYP317A对7‐乙氧基香豆素无活性。接下来，我们用黄酮类化合物黄酮作为底物，对这四个P450物种和之前表达的其他P450物种进行了筛选。结果，CYP2B、CYP2C43和CYP2C29催化黄酮2‐羟基化。CYP2A5催化2‐和4‐羟基化。最后，为了产生不同的修饰化合物，将具有点突变的CYP2A5变体与类固醇（DHEA）和抗氧化剂（黄酮）在体内孵育。HPLC分析表明，两个P450种产生7‐β‐羟基脱氢表雄酮，两个P450种产生2‐α‐羟基脱氢表雄酮。四种P450催化黄酮2 -和4 -羟基化。这些结果表明，P450的生物转化是一种有用的技术，可以修饰小分子（类固醇、香豆素和黄酮），并产生新的、多样化的羟基化化合物，这可以用于药物发现的高通量筛选。
P450 (cytochrome P450) enzymes catalyse the mono‐oxygenation of a wide range of compounds such as steroids, fatty acids, vitamins and drugs. In the present paper we demonstrate a system for bioconverting diverse compounds [flavanone, DHEA (dehydroepiandrosterone) and 7‐ethoxycoumarin] using P450 species expressed in Escherichia coli. First, we expressed four P450 species: rabbit CYP2B (P450 family 2, subfamily B), fruitfly (Drosophila) CYP317A, rat CYP3A23 and mouse CYP2J5. Next, we added substrates directly to the incubation medium. The resulting metabolites were extracted and analysed by HPLC and spectrofluorimetry. The first substrate, 7‐ethoxycoumarin, was de‐ethylated by CYP2B; CYP2J5 and CYP3A23 showed weak activity, and CYP317A had no activity for 7‐ethoxycoumarin. We next used flavanone, a flavonoid, as a substrate for these four P450 species and other P450 species expressed previously. As a result, CYP2B, CYP2C43 and CYP2C29 catalysed flavanone 2‐hydroxylation. CYP2A5 catalysed 2‐ and 4‐hydroxylations. Finally, to produce diverse modified compounds, variants of CYP2A5 with point mutations were incubated with a steroid (DHEA) and an antioxidant (flavanone) in vivo. HPLC analysis indicated that two P450 species produced a 7‐β‐hydroxy‐DHEA and two P450 species produced a 2‐α‐hydroxy‐DHEA. Four P450 species catalysed flavanone 2‐ and 4‐hydroxylations. These results indicate that bioconversion by P450 is a useful technique to modify small molecules (steroids, coumarin and flavanone) and produce new, diverse hydroxylated compounds, which could be used for high‐throughput screening for drug discovery.