Hydrophilicity-Based Engineering of the Active Pocket of D-Amino Acid Oxidase Leading to Highly Improved Specificity toward D-Glufosinate
Kai Yang
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorBin Huang
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorCharles Amanze
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorZhen Yan
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorGuanzhou Qiu
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorProf. Xueduan Liu
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorProf. Hongbo Zhou
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorCorresponding Author
Prof. Weimin Zeng
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorKai Yang
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorBin Huang
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorCharles Amanze
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorZhen Yan
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorGuanzhou Qiu
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorProf. Xueduan Liu
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorProf. Hongbo Zhou
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorCorresponding Author
Prof. Weimin Zeng
Central South University, School of Minerals Processing and Bioengineering, 932 South Lushan Road, Yuelu District, Changsha Hunan, 410083 P.R. China
Search for more papers by this authorAbstract
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.
Conflict of interest
The authors declare no conflict of interest.
Open Research
Data Availability Statement
The data that support the findings of this study are available in the supplementary material of this article.
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