Department of Chemistry, University of Rochester, Rochester, New York 14627, United States.
J Am Chem Soc. 2020 Jun 10;142(23):10343-10357. doi: 10.1021/jacs.9b12859. Epub 2020 Jun 1.
Cytochromes P450 have been recently identified as a promising class of biocatalysts for mediating C-H aminations via nitrene transfer, a valuable transformation for forging new C-N bonds. The catalytic efficiency of P450s in these non-native transformations is however significantly inferior to that exhibited by these enzymes in their native monooxygenase function. Using a mechanism-guided strategy, we report here the rational design of a series of P450-based variants with dramatically enhanced C-H amination activity acquired through disruption of the native proton relay network and other highly conserved structural elements within this class of enzymes. This approach further guided the identification of XplA and BezE, two "atypical" natural P450s implicated in the degradation of a man-made explosive and in benzastatins biosynthesis, respectively, as very efficient C-H aminases. Both XplA and BezE could be engineered to further improve their C-H amination reactivity, which demonstrates their evolvability for abiological reactions. These engineered and natural P450 catalysts can promote the intramolecular C-H amination of arylsulfonyl azides with over 10 000-14 000 catalytic turnovers, ranking among the most efficient nitrene transfer biocatalysts reported to date. Mechanistic and structure-reactivity studies provide insights into the origin of the C-H amination reactivity enhancement and highlight the divergent structural requirements inherent to supporting C-H amination versus C-H monooxygenation reactivity within this class of enzymes. Overall, this work provides new promising scaffolds for the development of nitrene transferases and demonstrates the value of mechanism-driven rational design as a strategy for improving the catalytic efficiency of metalloenzymes in the context of abiological transformations.
细胞色素 P450 最近被鉴定为一类有前途的生物催化剂，可通过氮烯转移介导 C-H 胺化反应，这是一种形成新 C-N 键的有价值的转化。然而，P450 在这些非天然转化中的催化效率明显低于其在天然单加氧酶功能中表现出的效率。在这里，我们使用一种基于机制的策略，报告了一系列基于 P450 的变体的合理设计，这些变体通过破坏天然质子传递网络和该酶类中的其他高度保守的结构元件，显著提高了 C-H 胺化活性。这种方法进一步指导了鉴定 XplA 和 BezE，这两种分别参与人造爆炸物降解和苯并他汀生物合成的“非典型”天然 P450，作为非常有效的 C-H 胺化酶。XplA 和 BezE 都可以被工程化以进一步提高其 C-H 胺化反应性，这证明了它们在非生物反应中的可进化性。这些工程化和天然的 P450 催化剂可以促进芳基磺酰基叠氮化物的分子内 C-H 胺化，催化周转率超过 10,000-14,000，是迄今为止报道的最有效的氮烯转移生物催化剂之一。机理和结构-反应性研究提供了对 C-H 胺化反应性增强的起源的深入了解，并突出了支持此类酶中 C-H 胺化与 C-H 单加氧酶反应性的固有不同结构要求。总的来说，这项工作为氮烯转移酶的发展提供了新的有前途的支架，并证明了基于机制的合理设计作为提高金属酶在非生物转化背景下催化效率的策略的价值。
