Rational Redesign of Enzyme via the Combination of Quantum Mechanics/Molecular Mechanics, Molecular Dynamics, and Structural Biology Study
Hong‐Yan Lin, Xi Chen, Jin Dong, Jing‐Fang Yang, Han Xiao, Ying Ye, Linhui Li, Chang‐Guo Zhan, Wen‐Chao Yang, Guang‐Fu Yang
Abstract
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 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.