Mutating a Highly Conserved Residue in Diverse Cytochrome P450s Facilitates Diastereoselective Olefin Cyclopropanation.
Mutating a Highly Conserved Residue in Diverse Cytochrome P450s Facilitates Diastereoselective Olefin Cyclopropanation.
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DOI：
10.1002/cbic.201500624
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发表时间：
2016-03-02
期刊：
影响因子：
--
通讯作者：
Brustad EM
中科院分区：
文献类型：
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作者：
Gober JG;Rydeen AE;Gibson-O'Grady EJ;Leuthaeuser JB;Fetrow JS;Brustad EM
细胞色素P450和其他含血红素的蛋白质最近已被证明具有混杂的活性，使用重氮乙酸试剂的烯烃环丙烷化。尽管迄今为止取得了进展，但为环丙烷化反应的所有可能的立体异构体设计选择性催化剂仍然是一个相当大的挑战。先前对模型P450（P450BM 3）的研究表明，保守活性位点苏氨酸（Thr268）突变为丙氨酸将酶转化为高活性和选择性的环丙烷化催化剂。通过将这种突变到一个不同的面板的P450支架，我们能够快速识别所有可能的非对映异构体的模型反应中的苯乙烯与重氮乙酸乙酯的对映选择性催化剂。一些丙氨酸变体表现出与野生型酶明显不同的选择性，其中一些具有中等至高的非对映体选择性，并且对于合成挑战性顺式环丙烷非对映体的对映体选择性高达97%。
Cytochrome P450s and other heme-containing proteins have recently been shown to have promiscuous activity for the cyclopropanation of olefins using diazoacetate reagents. Despite the progress made thus far, engineering selective catalysts for all possible stereoisomers for the cyclopropanation reaction remains a considerable challenge. Previous investigations of a model P450 (P450BM3) revealed that mutation of a conserved active site threonine (Thr268) to alanine transformed the enzyme into a highly active and selective cyclopropanation catalyst. By incorporating this mutation into a diverse panel of P450 scaffolds, we were able to quickly identify enantioselective catalysts for all possible diastereomers in the model reaction of styrene with ethyl diazoacetate. Some alanine variants exhibited selectivities that were markedly different from the wild-type enzyme, with a few possessing moderate to high diastereoselectivity and enantioselectivities up to 97 % for synthetically challenging cis-cyclopropane diastereomers.