Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 143, Issue 38, Pages 15674-15687Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.1c06227
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Funding
- National Key Research and Development Program of China [2017YFD0200500]
- National Natural Science Foundation of China [21837001, 22007035, U20A2038, 21273089]
- Hubei Province Natural Science Foundation [2020BHB027, 2020CFB487]
- Fundamental Research Funds for the South-Central University for Nationalities [CZW20020]
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The study introduces a rational redesign strategy for enzyme engineering based on QM/MM calculation and molecular dynamic simulations, leading to the discovery of new HPPD mutants and products. The findings not only enhance understanding of the HPPD catalytic pathway but also demonstrate a generally applicable platform for biocatalyst development.
Increasing demands for efficient and versatile chemical reactions have prompted innovations in enzyme engineering. A major challenge in engineering alpha-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 Arabidopsis thaliana 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.
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