Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 61, Issue 46, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202212720
Keywords
D-Amino Acid Oxidase; Glufosinate; Hydrophilicity; Protein Engineering; Triple-Code Saturation Mutagenesis
Categories
Funding
- National Natural Science Foundation of China [51934009, 52074353]
- National Key Research and Development Program of China [2019YFC1803600]
- Natural Science Foundation of Hunan Province [2021JJ30855, 2021JJ30836]
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In this study, the catalytic activity of DAAO towards D-glufosinate (D-PPT) was significantly increased through mutagenesis, leading to the establishment of an efficient enzymatic route for synthesizing L-PPT. This research also proposes a new perspective on enzyme engineering for the biosynthesis of unnatural amino acids.
Due to its stringent stereospecificity, D-amino acid oxidase (DAAO) has made it very easy to synthesize L-amino acids. However, the low activity of the wild-type enzyme toward unnatural substrates, such as D-glufosinate (D-PPT), restricts its application. In this study, DAAO from Rhodotorula gracilis (RgDAAO) was directly evolved using a hydrophilicity-substitution saturation mutagenesis strategy, yielding a mutant with significantly increased catalytic activity against D-PPT. The mutant displays distinct catalytic properties toward hydrophilic substrates as compared to numerous WT-DAAOs. The analysis of homology modeling and molecular dynamic simulation suggest that the extended reaction pocket with greater hydrophilicity was the reason for the enhanced activity. The current study established an enzymatic synthetic route to L-PPT, an excellent herbicide, with high efficiency, and the proposed strategy provides a new viewpoint on enzyme engineering for the biosynthesis of unnatural amino acids.
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