Carminati Daniela M, Moore Eric J, Fasan Rudi
Department of Chemistry, University of Rochester, Rochester, NY, United States.
Department of Chemistry, University of Rochester, Rochester, NY, United States.
Methods Enzymol. 2020;644:35-61. doi: 10.1016/bs.mie.2020.07.007. Epub 2020 Aug 6.
Myoglobin has recently emerged as a versatile metalloprotein scaffold for the design of efficient and selective biocatalysts for abiological carbene transfer reactions, including asymmetric cyclopropanation reactions. Over the past few years, our group has explored several strategies to modulate the carbene transfer reactivity of myoglobin-based catalysts, including the substitution of the native heme cofactor and conserved histidine axial ligand with non-native porphynoid ligands and alternative natural and unnatural amino acids as the metal-coordinating ligands, respectively. Herein, we report protocols for the generation and reconstitution in vitro and in vivo of myoglobin-based artificial carbene transferases incorporating non-native iron-porphynoid cofactors, also in combination with unnatural amino acids as the proximal ligand. These strategies are effective for imparting these myoglobin-based cyclopropanation biocatalysts with altered and improved function, including tolerance to aerobic conditions and improved reactivity toward electrondeficient olefins.
肌红蛋白最近已成为一种多功能金属蛋白支架，用于设计高效且选择性的生物催化剂，以进行包括不对称环丙烷化反应在内的非生物卡宾转移反应。在过去几年中，我们团队探索了多种策略来调节基于肌红蛋白的催化剂的卡宾转移反应活性，包括分别用非天然卟啉类配体替代天然血红素辅因子和保守的组氨酸轴向配体，以及用替代的天然和非天然氨基酸作为金属配位配体。在此，我们报告了在体外和体内生成并重组基于肌红蛋白的人工卡宾转移酶的方案，这些酶包含非天然铁卟啉类辅因子，也可与非天然氨基酸作为近端配体结合使用。这些策略对于赋予这些基于肌红蛋白的环丙烷化生物催化剂改变且改善的功能是有效的，包括对有氧条件的耐受性以及对缺电子烯烃的反应活性提高。
