Through virtual saturation mutagenesis and rational design for superior substrate conversion in engineered d-amino acid oxidase.
Biotechnol J
; 19(7): e2400287, 2024 Jul.
Article
em En
| MEDLINE
| ID: mdl-39014925
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Engenharia de Proteínas
/
D-Aminoácido Oxidase
Idioma:
En
Revista:
Biotechnol J
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2024
Tipo de documento:
Article