Knight Anders M, Kan S B Jennifer, Lewis Russell D, Brandenberg Oliver F, Chen Kai, Arnold Frances H
Division of Biology and Bioengineering and Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, MC 210-41, Pasadena, California 91125, United States.
ACS Cent Sci. 2018 Mar 28;4(3):372-377. doi: 10.1021/acscentsci.7b00548. Epub 2018 Feb 21.
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 cells in mild, aqueous conditions and provide the first example of enantioselective, intermolecular iron-catalyzed cyclopropanation of unactivated alkenes.
开发能够选择性地生成目标产物的每种立体异构体的催化剂是一项长期存在的合成挑战。生物化学家通过筛选自然界的多样性来应对这一挑战，以发现催化互补立体异构体形成的酶。我们在此表明，相同的方法可应用于一种新的天然酶促反应，即通过卡宾转移进行烯烃环丙烷化反应。通过筛选各种天然和工程化的血红素蛋白，我们鉴定出血红蛋白以及细胞色素P450的丝氨酸连接的“P411”变体，它们对未活化的烯烃底物进行环丙烷化反应具有混杂活性。然后，我们通过定向进化提高了它们的活性和立体选择性：仅进行1至3轮位点饱和诱变和筛选，就产生了能将未活化的烯烃和缺电子烯烃转化为四种立体异构环丙烷中的每一种的酶，总周转数高达5400，对映体过量率达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全细胞中发挥作用，并提供了未活化烯烃对映选择性、分子间铁催化环丙烷化反应的首个实例。
