Structure-guided engineering of substrate specificity in yeast d-aspartate oxidase

Selective quantification of individual d-amino acids is crucial for functional studies and biomarker development. Enzymatic assays enable convenient and rapid detection of d-amino acids. d-Amino acid oxidases (DAAOs) and d-aspartate oxidases (DDOs) are widely used for such assays; however, their broad substrate specificity limits selective detection in mixed samples. Here, we identified determinants of substrate recognition in DDO from the yeast Vanrija humicola (ChDDO) and reprogrammed its substrate specificity by structure-guided engineering. Crystal structure analysis highlighted Arg243 at the active-site entrance, and mutational analysis confirmed that substitution of Arg243 abolished detectable activity toward d-Asp and uncovered activity toward d-His and d-Phe. Molecular dynamics simulations of ChDDO indicated that Arg243 preferentially samples an inward-facing conformation toward His56 relative to the crystal-like outward orientation, accompanied by local rearrangements, suggesting a dynamic d-Asp uptake mechanism involving Arg243. Using R243A as a scaffold, we introduced second-site mutations to refine substrate specificity: L58N/R243A and F60Q/R243A increased d-His selectivity primarily by decreasing the apparent k_cat for d-Phe, whereas H56E/R243A increased d-Phe selectivity primarily by decreasing the apparent k_cat for d-His and increasing the apparent k_cat for d-Phe. The engineered variants enabled quantification of d-His or d-Phe in various media, even in the presence of a mixture of 17 non-target d-amino acids. This study suggests a potentially distinct gating mechanism in ChDDO and provides a structure-guided route to convert strict DDOs into tailored biocatalysts for analytical and biotechnological applications.