Site-directed mutagenesis as a tool for molecular modeling of cytochrome P450 2B1.
Site-directed mutagenesis as a tool for molecular modeling of cytochrome P450 2B1.
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DOI：
10.1021/bi00044a008
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发表时间：
1995-11
期刊：
影响因子：
2.9
通讯作者：
G. Szklarz;Y. He;J. Halpert
中科院分区：
文献类型：
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作者：
G. Szklarz;Y. He;J. Halpert
通过我们先前基于P450 cam的3-D结构的细胞色素P450 2B 1的同源模型[Szklarz，G. D、奥恩施泰因河L.，& Halpert，J. R.等人（1994）J. Biomol.结构动力学12，61-78]，我们在100，111，205，209，291，477和480位构建了11个新的定点突变体，并在大肠杆菌中表达了这些酶。位置209、477和480处的突变影响雄烯二酮和孕酮羟基化，如模型所预测。例如，Ile-477-->Ala和Ile-480-->Ala突变体保留了Ala突变体催化孕酮的21-羟基化。其他位置的突变，即，100，111，205和291，没有改变酶的活性，与预测相反。因此，我们建立了一个改进的细胞色素P450 2B 1的分子模型。P450 2B 1序列与P450 BM-3，P450 cam和P450 terp的比对使用来自各种细胞色素P450 2B中27个位置的定点诱变和雄烯二酮对接到已知晶体结构的活性位点的数据进行优化。由于发现所有三种结构都是P450 2B 1的合适模板，因此使用共识策略（一种基于距离几何计算的建模方法）基于三种蛋白质的晶体学坐标制定了新模型。新的模型提供了一种手段来解释在关键氨基酸位置，包括位置114，206，209，290，302，363，367，477，478和480在P450 2B 1的残基取代类固醇羟基化的区域和立体特异性的改变。
Prompted by our previous homology model of cytochrome P450 2B1 based on the 3-D structure of P450cam [Szklarz, G. D., Ornstein, R. L., & Halpert, J. R. (1994) J. Biomol. Struct. Dyn. 12, 61-78], we constructed 11 new site-directed mutants at positions 100, 111, 205, 209, 291, 477, and 480 and expressed the enzymes in Escherichia coli. The mutations at positions 209, 477, and 480 affected androstenedione and progesterone hydroxylation as predicted by the model. For example, the Ile-477-->Ala and Ile-480-->Ala mutants retained Ala mutant catalyzed 21-hydroxylation of progesterone. Mutations at the other positions, i.e., 100, 111, 205, and 291, did not change enzyme activity, contrary to predictions. Therefore, an improved molecular model of cytochrome P450 2B1 was constructed. An alignment of the P450 2B1 sequence with P450 BM-3, P450cam, and P450terp was optimized using data from site-directed mutagenesis at 27 positions in various cytochromes P450 2B and docking of androstenedione into the active site of the known crystal structures. Because all three structures were found to be suitable templates for P450 2B1, the new model was formulated on the basis of the crystallographic coordinates of the three proteins using a consensus strategy, a modeling method based on distance geometry calculations. The new model provides a means to explain alterations in regio- and stereospecificity of steroid hydroxylation upon residue substitution at key amino acid positions, including positions 114, 206, 209, 290, 302, 363, 367, 477, 478, and 480 in P450 2B1.