Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint Research Center for Intelligent Biosensor Technology and Health, College of Chemistry, Central China Normal University, Wuhan 430079, P.R. China.
College of Chemistry and Material Science, South-Central University for Nationalities, Wuhan 430074, P.R. China.
J Am Chem Soc. 2021 Sep 29;143(38):15674-15687. doi: 10.1021/jacs.1c06227. Epub 2021 Sep 20.
Increasing demands for efficient and versatile chemical reactions have prompted innovations in enzyme engineering. A major challenge in engineering α-ketoglutarate-dependent oxygenases is to develop a rational strategy which can be widely used for directly evolving the desired mutant to generate new products. Herein, we report a strategy for rational redesign of a model enzyme, 4-hydroxyphenylpyruvate dioxygenase (HPPD), based on quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamic simulations. This strategy enriched our understanding of the HPPD catalytic reaction pathway and led to the discovery of a series of HPPD mutants producing hydroxyphenylacetate (HPA) as the alternative product other than the native product homogentisate. The predicted HPPD-Fe(IV)═O-HPA intermediate was further confirmed by the crystal structure of HPPD/S267W complexed with HPA. These findings not only provide a good understanding of the structure-function relationship of HPPD but also demonstrate a generally applicable platform for the development of biocatalysts.
不断增长的对高效和多功能化学反应的需求促使酶工程领域的创新。在工程化α-酮戊二酸依赖的加氧酶方面的一个主要挑战是开发一种可广泛用于直接进化所需突变体以产生新产品的合理策略。在此，我们报道了一种基于量子力学/分子力学(QM/MM)计算和分子动力学模拟的模型酶 4-羟基苯丙酮酸双加氧酶(HPPD)的合理重新设计策略。该策略丰富了我们对 HPPD 催化反应途径的理解，并发现了一系列产生羟苯基乙酸酯(HPA)作为替代产物而不是天然产物反式粘康酸的 HPPD 突变体。通过与 HPA 结合的 HPPD/S267W 复合物的晶体结构进一步证实了预测的 HPPD-Fe(IV)═O-HPA 中间体。这些发现不仅提供了对 HPPD 结构-功能关系的深入了解，而且还展示了用于开发生物催化剂的通用平台。
