P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency
P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency
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DOI：
10.1093/jb/mvaa083
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发表时间：
2020-11-01
影响因子：
2.7
通讯作者：
Fukui，Kiyoshi
中科院分区：
文献类型：
--
作者：
Rachadech，Wanitcha;Kato，Yusuke;Fukui，Kiyoshi
人D-氨基酸氧化酶（DAO）是一种与神经退行性疾病有关的黄素酶。我们研究了在人DAO的活性位点lid中的位置219（P219 L）处用亮氨酸替换脯氨酸对结构和酶性质的影响，因为猪DAO在相应位置处含有亮氨酸。与野生型相比，P219 L的转化数（kcat）没有变化，但Km值降低，导致催化效率（kcat/Km）增加。此外，与野生型（1.2-2.0 µM）相比，苯甲酸盐以较低的Ki值（0.7-0.9 µ M）抑制P219 L。P219 L与黄素腺嘌呤二核苷酸（FAD）和苯甲酸盐复合物的晶体结构在2.25 nm分辨率下显示活性位点和盖的构象变化。精氨酸283和苯甲酸酯的氢键形成原子之间的距离以及酪氨酸224和苯甲酸酯的芳环之间的相对位置在P219 L复合物中发生了变化。总之，由于这些结构变化，P219 L取代导致催化效率和对底物/抑制剂的结合亲和力增加。此外，在P219 L复合物中，乙酸位于FAD的腺嘌呤环附近。这项研究为人类DAO的结构-功能关系提供了新的见解。
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 itsKmvalues decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lowerKivalue (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.