posted on 2021-09-20, 16:07authored byHong-Yan Lin, Xi Chen, Jin Dong, Jing-Fang Yang, Han Xiao, Ying Ye, Lin-Hui Li, Chang-Guo Zhan, Wen-Chao Yang, Guang-Fu Yang
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.