Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability
Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability
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DOI：
10.1074/jbc.ra119.010727
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发表时间：
2020-04-24
影响因子：
4.8
通讯作者：
Scott, Emily E.
中科院分区：
文献类型：
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作者：
Bart, Aaron G.;Harris, Kurt L.;Scott, Emily E.
哺乳动物细胞色素P450酶经常代谢许多药物和其他外源物质，这一特征在生物技术环境中很有价值。然而，现有的P450酶通常是相对不稳定的，T-50值为30？40℃。与现有的相关形式相比，重构的祖先细胞色素P450酶往往具有不同的底物选择性，但它们也具有更高的热稳定性，因此可能是重要中间体、最终药物分子或药物代谢物的商业生物合成的优秀工具。哺乳动物细胞色素P4501B亚家族的祖先在结构和功能上与现存的人类细胞色素P4501B1在配体结合、代谢和热稳定性的潜在分子贡献方面存在差异。虽然现存的人CYP1B1在一个封闭的活性部位有一个β-萘黄酮分子，但我们观察到在祖先的CYP1B酶活性部位外的细微氨基酸替换产生了一个开放的活性部位，有四个配基副本。含有17？-雌二醇的祖先的结构显示，活性部位只有一个分子，仍然具有相同的开放构象。对现存形式和祖先形式的详细比较表明，祖先形式中不同的二级结构元素之间的静电和芳香相互作用增加，这可能有助于它们的热稳定性。据我们所知，这是第一次对重建的祖先细胞色素P450进行结构评估，揭示了似乎有助于其热稳定性的关键特征。
Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T-50 values of ?30?40 ?C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of ?-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17?-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.