Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585 (Singapore) http://www.chbe.nus.edu.sg/people/faculty_liz.html.
Angew Chem Int Ed Engl. 2014 Mar 17;53(12):3120-4. doi: 10.1002/anie.201311091. Epub 2014 Feb 19.
Terminal-selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio- and enantioselectivity. A sensitive colorimetric high-throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple-mutant P450pyrSM1 was created for the hydroxylation of n-octane (1) to give (S)-2-octanol (2) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C-H activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure-based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple-mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene (3) to afford (S)-1-phenyl-2-propanol (4) with 95 % ee and 98 % subterminal selectivity.
通过定向进化成功地工程改造了末端选择性细胞色素 P450pyr，使其能够对烷烃进行亚末端羟化，具有优异的区域和对映选择性。开发了一种灵敏的比色高通量筛选 (HTS) 测定法，用于测量羟化反应的区域选择性和对映选择性。通过使用 HTS 测定法和迭代饱和突变，创建了六重突变体 P450pyrSM1，用于将正辛烷 (1) 羟化生成 (S)-2-辛醇 (2)，ee 值为 98%，亚末端选择性>99%。该工程化的 P450 是用于这种高度选择性烷烃羟化的第一种酶，可用于烷烃的 C-H 活化和官能化以及手性纯醇的制备。分子建模提供了基于结构的完全改变的区域选择性和优异的对映选择性的理解。另一个六重突变体 P450pyrSM2 催化了丙基苯 (3) 的羟化，得到 (S)-1-苯基-2-丙醇 (4)，ee 值为 95%，亚末端选择性为 98%。
