P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping
P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping
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DOI：
10.1021/acscatal.8b00389
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发表时间：
2018-04-01
期刊：
影响因子：
12.9
通讯作者：
Reetz, Manfred T.
中科院分区：
文献类型：
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作者：
Acevedo-Rocha, Carlos G.;Gamble, Charles G.;Reetz, Manfred T.
细胞色素P450单加氧酶在许多天然产物的生物合成和许多药物的人体代谢中发挥着至关重要的作用。这激发了合成有机和药物化学家利用它们作为催化剂在区域和立体选择性CH-活化氧化的结构简单和复杂的有机化合物，如类固醇。然而，对于真实的应用，区域和立体选择性以及活性的水平通常不够高。使用合理设计或定向进化的蛋白质工程在许多方面都有帮助，但同时工程化多种催化特性，如活性，区域选择性和立体选择性，同时克服权衡和收益递减，仍然是一个挑战。在这里，我们表明，利用来自突变景观和分子动力学模拟合理设计迭代饱和诱变的信息构成了一个可行的定向进化策略。P450（BM 3）突变体的进化说明了这种组合的方法，所述突变体能够使五种不同的甾族化合物特别是在C16-位置具有异常高的活性的几乎完美的区域选择性和非对映选择性羟基化，同时避免活性-选择性权衡以及保持筛选工作相对较低。C16醇作为生物活性糖皮质激素的组分具有实际意义。
Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio- and stereoselective CH-activating oxidation of structurally simple and complex organic compounds such as steroids. However, levels of regio- and stereoselectivity as well as activity are not routinely high enough for real applications. Protein engineering using rational design or directed evolution has helped in many respects, but simultaneous engineering of multiple catalytic traits such as activity, regioselectivity, and stereoselectivity, while overcoming trade-offs and diminishing returns, remains a challenge. Here we show that the exploitation of information derived from mutability landscapes and molecular dynamics simulations for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy. This combined approach is illustrated by the evolution of P450(BM3) mutants which enable nearly perfect regio- and diastereoselective hydroxylation of five different steroids specifically at the C16-position with unusually high activity, while avoiding activity-selectivity trade-offs as well as keeping the screening effort relatively low. The C16 alcohols are of practical interest as components of biologically active glucocorticoids.