Enhancement of the substrate specificity of D‐amino acid oxidase based on tunnel‐pocket engineering

Abstract D ‐Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of D ‐amino acids, making it one of the most promising routes for synthesizing optically pure L ‐amino acids, including L ‐phosphinothricin ( L ‐PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate D ‐PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis ( Rtw DAAO), and improved its catalytic potential toward D ‐PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel‐pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M 3rd ‐SHVG was obtained, exhibiting a >2000‐fold increase in relative activity. The kinetic parameters showed that M 3rd ‐SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M 3rd ‐SHVG reshapes the tunnel‐pocket and corrects the direction of enzyme–substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel‐pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M 3rd ‐SHVG as biocatalyst, 500 mM D, L ‐PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.