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首页> 外文期刊>Biochemistry >SITE-DIRECTED MUTAGENESIS AS A TOOL FOR MOLECULAR MODELING OF CYTOCHROME P450 2B1
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SITE-DIRECTED MUTAGENESIS AS A TOOL FOR MOLECULAR MODELING OF CYTOCHROME P450 2B1

机译：现场定向诱变作为细胞色素P450 2B1分子建模的工具

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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. Srrrtct. Dyn. 12, 61-78], we constructed ii new site-directed mutants at positions 100, 111, 205, 209, 291, 477, and 480 and expressed the enzymes in Escherichia coil. 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 less than or equal to 5% activity with androstenedione and progesterone but were active with benzphetamine, whereas the Leu-209-->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. [References: 55]
机译：由我们先前基于P450cam的3-D结构的细胞色素P450 2B1同源模型提示[Szklarz，G.D.，Ornstein，R.L.＆Halpert，J.R.（1994）J. Biomol。简写。达因12，12，61-78]，我们在位置100、111、205、209、291、477和480处构建了新的定点突变体，并在大肠杆菌中表达了酶。如模型所预测的，第209、477和480位的突变影响了雄烯二酮和孕酮的羟基化。例如，Ile-477-> Ala和Ile-480-> Ala突变体与雄烯二酮和孕酮的活性保持小于或等于5％，但对苯丙胺具有活性，而Leu-209-> Ala突变体被催化孕酮的21-羟基化。与预测相反，在其他位置，即100、111、205和291处的突变并未改变酶的活性。因此，构建了细胞色素P450 2B1的改进分子模型。 P450 2B1序列与P450 BM-3，P450cam和P450terp的比对使用来自各种细胞色素P450 2B中27个位置的定点诱变数据和雄烯二酮对接到已知晶体结构的活性位点的数据进行了优化。由于发现所有这三种结构都是适用于P450 2B1的模板，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来制定新模型。新模型提供了一种手段，用于解释在关键氨基酸位置（包括P450中的114、206、209、290、302、363、367、477、477、478和480位）进行残基取代时类固醇羟基化的区域和立体特异性的变化2B1。 [参考：55]

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