Substrate-Oriented Channel Engineering of D-Amino Acid Oxidase for Efficient L/D-Phosphinothricin Resolution: Synergies of Geometric and Electrostatic Modifications

D-Amino acid oxidase (DAAO) catalyzes the oxidation of D-phosphinothricin (D-PPT) to produce optically pure L-PPT, but low catalytic efficiency limits its industrial application. Here, a synergistic strategy combining geometric remodeling and charge engineering was applied to enhance the oxidation activity of our previously developed stable variant TIF-DAAO (S18T/V7I/Y132F) toward D-PPT. Systematic analysis of the active pocket-guided alanine and neutral hydrophilic (Ser/Thr/Tyr) scanning mutagenesis revealed position-specific steric constraints. Subsequent Arg/His scanning tailored the positively charged microenvironment around D-PPT, leading to a triple mutant ATR (F58A/Q335T/P221R) with improved electrostatic complementarity and spatial fit. ATR showed a 10.7-fold increase in catalytic activity, a 35-fold improvement in substrate affinity, and a 380-fold higher catalytic efficiency ( k cat / K M ) toward D-PPT. Molecular dynamics simulations provided mechanistic insight. Our results demonstrate that the synergistic optimization of substrate tunnel geometry and the electrostatic microenvironment effectively boosts DAAO activity, offering a rational strategy for engineering industrial biocatalysts.