Crystal structure of human D-amino acid oxidase:: Context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring
Crystal structure of human D-amino acid oxidase:: Context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring
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DOI：
10.1110/ps.062421606
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发表时间：
2006-12-01
期刊：
影响因子：
8
通讯作者：
Fukui, Kiyoshi
中科院分区：
文献类型：
--
作者：
Kawazoe, Tomoya;Tsuge, Hideaki;Fukui, Kiyoshi
在大脑中，广泛研究的FAD依赖性酶D-氨基酸氧化酶（DAO）降解神经胶质递质D-丝氨酸，N-甲基-D-天冬氨酸型谷氨酸受体的有效激活剂，有证据表明，DAO及其激活剂G72蛋白，可能在精神分裂症的病理生理学中发挥关键作用。事实上，其潜在的临床重要性突出了对人类DAO的结构和功能分析的需要。我们最近成功地纯化了人DAO，并发现它与FAD的结合较弱，与猪DAO相比，黄素还原速率明显较慢。然而，不同动力学特征的分子基础仍不清楚，因为认为人DAO的活性位点与猪DAO的活性位点几乎相同，如85%序列同一性所预期。为了解决这个问题，我们确定了与竞争性抑制剂苯甲酸盐复合的人类DAO的晶体结构，分辨率为2.5埃。人DAO的总体二聚体结构与猪DAO相似，并且催化残基在黄素环的背面完全保守。然而，在黄素环的si面，尽管具有严格的序列同一性，但与两种独立确定的猪DAO-苯甲酸酯结构相比，疏水段（残基47-51，VAAGL）以显着不同的构象存在。这表明，疏水性拉伸的上下文依赖性构象变异性解释了对FAD的低亲和力以及黄素还原的较慢速率，从而突出了人类酶的独特特征。
In the brain, the extensively studied FAD-dependent enzyme D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent activator of N-methyl-D-aspartate type glutamate receptors, and evidence suggests that DAO, together with its activator G72 protein, may play a key role in the pathophysiology of schizophrenia. Indeed, its potential clinical importance highlights the need for structural and functional analyses of human DAO. We recently succeeded in purifying human DAO, and found that it weakly binds FAD and shows a significant slower rate of flavin reduction compared with porcine DAO. However, the molecular basis for the different kinetic features remains unclear because the active site of human DAO was considered to be virtually identical to that of porcine DAO, as would be expected from the 85% sequence identity. To address this issue, we determined the crystal structure of human DAO in complex with a competitive inhibitor benzoate, at a resolution of 2.5 angstrom. The overall dimeric structure of human DAO is similar to porcine DAO, and the catalytic residues are fully conserved at the re-face of the flavin ring. However, at the si-face of the flavin ring, despite the strict sequence identity, a hydrophobic stretch (residues 47-51, VAAGL) exists in a significantly different conformation compared with both of the independently determined porcine DAO-benzoate structures. This suggests that a context-dependent conformational variability of the hydrophobic stretch accounts for the low affinity for FAD as well as the slower rate of flavin reduction, thus highlighting the unique features of the human enzyme.