Structural determinants of unique substrate specificity of d-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii

d-Amino acid oxidase from the thermophilic fungus Rasamsonia emersonii (ReDAAO) has garnered attention due to its high stability and broad substrate specificity, making it a promising candidate for various applications. In this study, we explored the structural factors underlying the unique substrate specificity of ReDAAO, particularly its broad substrate range and d-Glu oxidation ability. Comparing ReDAAO with TdDAAO—a homologous d-amino acid oxidase from the thermophilic fungus Thermomyces dupontii—revealed that ReDAAO lacks the YVLQG loop present in TdDAAO, which exhibited narrower substrate specificity. Inserting the YVLQG loop into ReDAAO narrowed its substrate specificity to match TdDAAO, while deleting the sequence from TdDAAO broadened its substrate specificity, resembling ReDAAO. A TdDAAO structural model suggests that the YVLQG loop could interact with a spatially adjacent region covering the active site, distinct from the canonical active-site lid in DAAOs, creating steric hindrance that limits access to the catalytic pocket. Additionally, the unexpected activity of ReDAAO toward d-Glu appears to depend on Arg97 and Ser231, which could interact with d-Glu side chain. Alanine substitutions at these residues significantly reduced d-Glu activity, revealing that Arg97 is essential for catalytic turnover while Ser231 is critical for substrate binding. Together, these results suggest that the YVLQG loop together with the spatially adjacent region acts as a steric gate that modulates access to the catalytic pocket, and Arg97/Ser231 plays an important role in d-Glu. These findings deepen our understanding of the structure–function relationship of DAAO and provide a foundation for developing improved DAAO variants for industrial applications.