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Genetic Incorporation of Selenotyrosine Significantly Improves Enzymatic Activity of Agrobacterium radiobacter Phosphotriesterase
Author(s) -
An Xiaojing,
Chen Chao,
Wang Tianyuan,
Huang Aiping,
Zhang Dawei,
Han MingJie,
Wang Jiangyun
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000460
Subject(s) - enzyme kinetics , enzyme , mutant , catalytic efficiency , tyrosine , biochemistry , amino acid , chemistry , directed evolution , active site , stereochemistry , protein engineering , gene
Tyrosine plays important roles in many enzymes. To facilitate enzyme design, mechanistic studies and minimize structural perturbation in the active site, here we report the genetic incorporation of a novel unnatural amino acid selenotyrosine (SeHF), which has single‐atom replacement in comparison to tyrosine. The ar PTE‐( Agrobacterium radiobacter Phosphotriesterase) Tyr309SeHF mutant exhibits a significant 12‐fold increase in k cat and 3.2‐fold enhancement in k cat / K M at pH 7.0. Molecular dynamics simulations show that the SeHF309 mutation results in a conformational switch which opens up the product release pocket and increases the product release rate, thereby elevating the overall enzyme activity. Significant improvement of the catalytic efficiency at neutral pH by single unnatural amino acid (UAA) mutation broadens the application of this enzyme, and provides valuable insights to the mechanism. Our method represents a new approach for designing enzymes with enhanced activity.