Developing catalysts that produce each stereoisomer of a desired product selectively is a longstanding synthetic challenge. Biochemists have addressed this challenge by screening nature's diversity to discover enzymes that catalyze the formation of complementary stereoisomers. We show here that the same approach can be applied to a new-to-nature enzymatic reaction, alkene cyclopropanation via carbene transfer. By screening diverse native and engineered heme proteins, we identified globins and serine-ligated "P411" variants of cytochromes P450 with promiscuous activity for cyclopropanation of unactivated alkene substrates. We then enhanced their activities and stereoselectivities by directed evolution: just 1-3 rounds of site-saturation mutagenesis and screening generated enzymes that transform unactivated alkenes and electron-deficient alkenes into each of the four stereoisomeric cyclopropanes with up to 5,400 total turnovers and 98% enantiomeric excess. These fully genetically encoded biocatalysts function in whole Escherichia coli cells in mild, aqueous conditions and provide the first example of enantioselective, intermolecular iron-catalyzed cyclopropanation of unactivated alkenes.

中文翻译：

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多种工程血红素蛋白可实现未活化烯烃的立体发散环丙烷化。

选择性地产生所需产物的每种立体异构体的催化剂的开发是长期以来的合成挑战。生物化学家通过筛选自然界的多样性来发现催化互补立体异构体形成的酶，从而解决了这一难题。我们在这里表明，相同的方法可以应用于新的自然酶反应，通过卡宾转移进行烯烃环丙烷化。通过筛选各种天然和工程血红素蛋白，我们鉴定了球蛋白和丝氨酸连接的细胞色素P450的“ P411”变体，具有对未活化烯烃底物进行环丙烷化的混杂活性。然后，我们通过定向进化增强了它们的活性和立体选择性：仅需进行1-3轮位点饱和诱变和筛选，即可产生将未活化的烯烃和缺电子的烯烃转化为四种立体异构环丙烷中的每一种的酶，总转化率高达5,400，对映异构体过量98％。这些完全由基因编码的生物催化剂在温和的水性条件下在整个大肠杆菌细胞中发挥功能，并提供了对映选择性，分子间铁催化的未活化烯烃的环丙烷化的第一个实例。