The C-terminal peptide plays a role in the formation of an intermediate form during the transition between xanthine dehydrogenase and xanthine oxidase.
The C-terminal peptide plays a role in the formation of an intermediate form during the transition between xanthine dehydrogenase and xanthine oxidase.
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DOI：
10.1111/febs.13277
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发表时间：
2015-08
期刊：
影响因子：
--
通讯作者：
Nishino T
中科院分区：
文献类型：
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作者：
Nishino T;Okamoto K;Kawaguchi Y;Matsumura T;Eger BT;Pai EF;Nishino T
哺乳动物黄嘌呤氧化还原酶可以脱氢酶和氧化酶两种形式存在。两者之间的转换涉及哺乳、抗菌活性、再灌注损伤和越来越多的疾病等多种过程。我们构建了一种缺乏羧基末端氨基酸1316-1331的大鼠肝酶变体;它似乎呈现中间形式，表现出脱氢酶和氧化酶活性的混合物。纯化的变体蛋白即使在长时间二硫苏糖醇处理后仍保留约50-70%的氧化酶活性，支持先前的预测，即C末端区域在脱氢酶转化为氧化酶中起作用。在蛋白质变体的晶体结构中，大多数酶保持氧化酶构象。然而，在与高浓度的NADH孵育15分钟后，相应的X射线结构显示出脱氢酶型构象。另一方面，Cys 535和Cys 992之间的二硫键形成（这可以在去除二硫苏糖醇后的变体的晶体结构的电子密度图中清楚地看到）与完全转化为氧化酶平行进行，导致与接头肽的蛋白水解切割后观察到的结构变化相同的结构变化。这些结果表明，脱氢酶-氧化酶转化相当容易发生，并且C末端肽插入其亚基的活性位点空腔中稳定了脱氢酶形式。我们提出，在酶的C末端肽部分与其他蛋白质或细胞膜相互作用后，可以产生中间形式（例如在内皮细胞中）。在本研究中报道的四种晶体结构的坐标集和结构因子已存放在蛋白质数据库中，标识号为4 YRW，4 YTZ，4 YSW和4 YTY。
Mammalian xanthine oxidoreductase can exist in both dehydrogenase and oxidase forms. Conversion between the two is implicated in such diverse processes as lactation, anti‐bacterial activity, reperfusion injury and a growing number of diseases. We have constructed a variant of the rat liver enzyme that lacks the carboxy‐terminal amino acids 1316–1331; it appears to assume an intermediate form, exhibiting a mixture of dehydrogenase and oxidase activities. The purified variant protein retained ~ 50–70% of oxidase activity even after prolonged dithiothreitol treatment, supporting a previous prediction that the C‐terminal region plays a role in the dehydrogenase to oxidase conversion. In the crystal structure of the protein variant, most of the enzyme stays in an oxidase conformation. After 15 min of incubation with a high concentration of NADH, however, the corresponding X‐ray structures showed a dehydrogenase‐type conformation. On the other hand, disulfide formation between Cys535 and Cys992, which can clearly be seen in the electron density map of the crystal structure of the variant after removal of dithiothreitol, goes in parallel with the complete conversion to oxidase, resulting in structural changes identical to those observed upon proteolytic cleavage of the linker peptide. These results indicate that the dehydrogenase–oxidase transformation occurs rather readily and the insertion of the C‐terminal peptide into the active site cavity of its subunit stabilizes the dehydrogenase form. We propose that the intermediate form can be generated (e.g. in endothelial cells) upon interaction of the C‐terminal peptide portion of the enzyme with other proteins or the cell membrane. Coordinate sets and structure factors for the four crystal structures reported in the present study have been deposited in the Protein Data Bank under the identification numbers 4YRW, 4YTZ, 4YSW, and 4YTY.