P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

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 to 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 to 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. The present study provides new insights into the structure-function relationship of human DAO.

内容要旨・審査要旨・論文本文の公開
本論文は，著者Wanitcha Rachadechの学位論文として提出され，学位審査・授与の対象となっている。
This is a pre-copyedited, author-produced version of an article accepted for publication in The Journal of Biochemistry following peer review. The version of record Wanitcha Rachadech, Yusuke Kato, Rabab M Abou El-Magd, Yuji Shishido, Soo Hyeon Kim, Hirofumi Sogabe, Nobuo Maita, Kazuko Yorita, Kiyoshi Fukui, P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency, The Journal of Biochemistry, Volume 168, Issue 5, November 2020, Pages 557–567 is available online at: https://doi.org/10.1093/jb/mvaa083