Structural Snapshots of Yeast Alkyl Hydroperoxide Reductase Ahp1 Peroxiredoxin Reveal a Novel Two-cysteine Mechanism of Electron Transfer to Eliminate Reactive Oxygen Species
Structural Snapshots of Yeast Alkyl Hydroperoxide Reductase Ahp1 Peroxiredoxin Reveal a Novel Two-cysteine Mechanism of Electron Transfer to Eliminate Reactive Oxygen Species
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DOI：
10.1074/jbc.m112.357368
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发表时间：
2012-05-18
影响因子：
4.8
通讯作者：
Zhou, Cong-Zhao
中科院分区：
文献类型：
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作者：
Lian, Fu-Ming;Yu, Jiang;Zhou, Cong-Zhao
过氧化物酶（Peroxiredoxins，Prxs）是一种巯基特异性抗氧化蛋白，参与细胞信号转导通路，保护细胞免受活性氧的侵害。烷基过氧化氢还原酶Ahp 1属于Prx 5亚家族，是一个形成分子间二硫键的双半胱氨酸（2-Cys）Prx。酶促分析和生物信息学使我们能够重新分配过氧化物半胱氨酸（C-P）的Cys-62和解析半胱氨酸（C-R）的Cys-31，但不是以前报道的Cys-120。因此，Ahp 1代表在一级序列中解析半胱氨酸之后的第一个具有过氧化物半胱氨酸的2-Cys Prx。我们还发现，正协同性的底物叔丁基氢过氧化物结合到Ahp 1同型二聚体在希尔系数类似于2，这使得Ahp 1消除氢过氧化物在更高的效率。为了深入了解Ahp 1的催化循环结构，我们分别以2.40、2.91和2.10埃的分辨率确定了Ahp 1在氧化、还原和Trx 2复合形式中的晶体结构。氧化态与还原态的结构叠加显示了含有C-P和C-R的片段的显著构象变化。一个跨A型二聚体界面的分子间C-P-C-R二硫键将Ahp 1与其他典型的2-Cys Prxs区分开来。Ahp 1-Trx 2复合物的结构首次显示了电子如何从硫氧还蛋白转移到具有硫氧还蛋白样折叠的过氧化物酶。此外，结合酶促试验的定点诱变表明，在Lys-32的urmylation（共价结合到泛素相关的修饰剂Urm 1）后，Ahp 1的过氧化物酶活性将被改变。
Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C-P) to Cys-62 and the resolving cysteine (C-R) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of similar to 2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 angstrom resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C-P and C-R. An intermolecular C-P-C-R disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.