Abstract
Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.
The reaction of 8-mercaptoflavins and flavoproteins with sulfite. Evidence for the role of an active site arginine in D-amino acid oxidase.
