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 (RtwDAAO), 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 M3rd‐SHVG was obtained, exhibiting a >2000‐fold increase in relative activity. The kinetic parameters showed that M3rd‐SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M3rd‐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 M3rd‐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.

Correlation Analysis of Key Residue Sites between Computational-Aided Design Thermostability d-Amino Acid Oxidase and Ancestral Enzymes

“…The mobile phase consisted of acetonitrile and 50 mM (NH 2 ) HPO 4 containing 1‰ tetrabutylammonium bromide (12:88, v/v) at 40 °C, pH 3.8, running at a flow rate of 1.0 mL min –1 . The HPLC, MS, and NMR analyses of substrates ( d , l -PPT) and products (PPO and l -PPT) refer to the recent results. , All d -amino acids were rapidly detected by a Micro Amino Acid Content Assay Kit. All experiments were repeated 3 times in the same batch.…”

“…The PCR reaction system and conditions were consistent with those previously reported. 6 The commercial catalase from Micrococcus was purchased from Sigma-Aldrich (Shanghai, China). Unless otherwise stated, all other chemicals and reagents used in this study were analytical grade and were commercially available.…”

“…The HPLC, MS, and NMR analyses of substrates (D,L-PPT) and products (PPO and L-PPT) refer to the recent results. 6,22 All D-amino acids were rapidly detected by a Micro Amino Acid Content Assay Kit. All experiments were repeated 3 times in the same batch.…”

“…d -Amino acid oxidase (DAAO; EC:1.4.3.3) catalyzes the oxidation of d -amino acids to produce the corresponding α-keto acids, leading to the disassembly of chiral amino acids and the generation of key intermediates. , DAAO plays a crucial role in the synthesis of drugs, pesticides, and fine chemicals due to its broad substrate spectrum and high enantioselectivity. − In our previous study, Rtw DAAO from Rhodotorula taiwanensis was cloned and expressed. Following modification, the variant SHVG has been demonstrated to exhibit higher kinetic parameters toward the non-natural substrate d -phosphinothricin ( D -PPT) . However, the poor thermostability of Rtw DAAO limits its further industrial application.…”

“…Following modification, the variant SHVG has been demonstrated to exhibit higher kinetic parameters toward the non-natural substrate Dphosphinothricin (D-PPT). 6 However, the poor thermostability of RtwDAAO limits its further industrial application. Typically, stabilizing one aspect of a protein stabilizes other aspects.…”