Through virtual saturation mutagenesis and rational design for superior substrate conversion in engineered d-amino acid oxidase.

Autor: Tang H; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, P. R. China.; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, P. R. China., Zhu HL; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, P. R. China.; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, P. R. China., Zhao JQ; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, P. R. China.; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, P. R. China., Wang LY; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, P. R. China.; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, P. R. China., Xue YP; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, P. R. China.; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, P. R. China., Zheng YG; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, P. R. China.; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, P. R. China.
Jazyk: angličtina
Zdroj: Biotechnology journal [Biotechnol J] 2024 Jul; Vol. 19 (7), pp. e2400287.
DOI: 10.1002/biot.202400287
Abstrakt: The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure.
(© 2024 Wiley‐VCH GmbH.)
Databáze: MEDLINE
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