Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 angstrom resolution
Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 angstrom resolution
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DOI：
10.1016/s0969-2126(96)00101-3
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发表时间：
1996-08-15
期刊：
影响因子：
5.7
通讯作者：
Zubak, VM
中科院分区：
文献类型：
--
作者：
Kumar, V;Dooley, DM;Zubak, VM
背景资料：含铜胺氧化酶在需要自由基的反应中催化伯胺氧化脱氨为醛。这些酶在许多生物过程中很重要，包括细胞分化和生长、伤口愈合、解毒和信号传导。催化反应需要氧化还原辅因子，托帕醌（TPQ），这是由一个不变的酪氨酸残基的翻译后修饰。TPQ辅因子的生物合成和酶催化的反应都需要在活性位点存在铜原子。对大肠杆菌铜胺氧化酶的晶体结构进行了研究。结果：在2.2埃的分辨率下，解析并精细化了真核生物（豌豆苗）胺氧化酶（PSAO）的一级结构。晶体相来自于一个单一的磷钨酸衍生物。用分子置换法确定了钨原子在W-12团簇中的位置。大肠杆菌胺氧化酶作为搜索模型。该方法避免了搜索模型的偏见，并提供了对真核胺氧化酶的基本独立的看法。PSAO分子是同二聚体;每个亚基具有三个结构域。每个亚基的活性位点位于最大结构域的β-夹心的边缘附近，但不能从溶剂中接近。必需的活性位点铜原子由三个组氨酸侧链和两个水分子以近似正方锥体的排列配位，TPQ辅因子的所有原子都明确定义，到铜原子的最短距离近似为6埃。PSAO和ECAO之间存在相当大的结构同源性，来自两种结构的证据的组合表明，TPQ侧链是足够柔性的，以允许芳族基团围绕C β-C γ键旋转，并且相对于Cu原子在成键和非成键位置之间移动。分子表面还需要构象柔性，以允许底物进入溶剂无法接近的活性位点，正如使用自由基化学的酶所预期的那样。
Background: Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, wound healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported.Results: The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 Angstrom resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W-12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains, The active site of each subunil: lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement, All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being similar to 6 Angstrom.Conclusions: There is considerable structural homology between PSAO and ECAO, A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic group to rotate about the C beta-C gamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.