Achieving Regio- and Enantioselectivity of P450-Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure-Guided Directed Evolution
Achieving Regio- and Enantioselectivity of P450-Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure-Guided Directed Evolution
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DOI：
10.1002/cbic.201200244
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发表时间：
2012-07-09
期刊：
影响因子：
3.2
通讯作者：
Reetz, Manfred T.
中科院分区：
文献类型：
--
作者：
Agudo, Ruben;Roiban, Gheorghe-Doru;Reetz, Manfred T.
单加氧酶P450-BM3的定向进化利用结合位点及其附近的迭代饱和突变，使得环己烯-1-羧酸甲酯的氧化羟基化具有高度的区域和对映选择性。野生型P450-BM3对烯丙基3位的区域选择性为84%，对R醇的对映体选择性为34%。进化出了允许R选择性(>；95？%ee)或S选择性(>；95？%ee)的突变体，同时减少了其他氧化产物，从而使区域选择性最大化至>；93？%。控制底物与酶的比例对于获得最佳的和可重现的对映体选择性是必要的，这一观察结果在这些单加氧酶的未来蛋白质工程中是重要的。还设计了一种能够再生NADPH的大肠杆菌菌株，总体上简化了P450酶的定向进化。这些合成结果为随后的立体选择性和立体特异性的化学转化形成更复杂的化合物奠定了基础，从而说明了将转基因酶作为有机化学中的催化剂与传统化学方法相结合的可行性。
Directed evolution of the monooxygenase P450-BM3 utilizing iterative saturation mutagenesis at and near the binding site enables a high degree of both regio- and enantioselectivity in the oxidative hydroxylation of cyclohexene-1-carboxylic acid methyl ester. Wild-type P450-BM3 is 84?% regioselective for the allylic 3-position with 34?% enantioselectivity in favor of the R alcohol. Mutants enabling R selectivity (>95?% ee) or S selectivity (>95?% ee) were evolved, while reducing other oxidation products and thus maximizing regioselectivity to >93?%. Control of the substrate-to-enzyme ratio is necessary for obtaining optimal and reproducible enantioselectivities, an observation which is important in future protein engineering of these mono-oxygenases. An E. coli strain capable of NADPH regeneration was also engineered, simplifying directed evolution of P450 enzymes in general. These synthetic results set the stage for subsequent stereoselective and stereospecific chemical transformations to form more complex compounds, thereby illustrating the viability of combining genetically altered enzymes as catalysts in organic chemistry with traditional chemical methods.