The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins
The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins
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DOI：
10.1021/bi800477u
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发表时间：
2008-06-17
期刊：
影响因子：
2.9
通讯作者：
Leimkuehler, Silke
中科院分区：
文献类型：
--
作者：
Schmitz, Jennifer;Chowdhury, Mita Mullick;Leimkuehler, Silke
由于泛素激活和几种含硫辅助因子的生物合成机制相似，我们对人类Urm1和酿酒酵母Uba4蛋白进行了表征，它们的序列分别与MOCS2A和MOCS3非常相似，MOCS2A和MOCS3是人类钼辅助因子（Moco）生物合成所必需的两个蛋白。MOCS3同源物的系统发育分析表明，Uba4是酵母中MOCS3的同源物，因此是该生物中Moco生物合成途径的唯一剩余蛋白。由于人MOCS3和酵母Uba4的序列高度同源，我们纯化了Uba4蛋白，并对其催化活性进行了详细的表征。我们证明了Uba4的c端结构域与MOCS3一样具有罗丹斯活性，并且能够在体外将硫代硫酸盐中的硫转移到氰化物中。此外，我们能够与人Urm1和MOCS2A共纯化稳定的Uba4异四聚体复合物。Uba4的n端结构域通过形成酰基-腺苷酸键来催化MOCS2A或Urm1的激活。经过腺苷化后，过硫Uba4能够在Urm1或MOCS2A的c端甘氨酸上形成硫代羧酸基。未观察到Uba4与Urm1或MOCS2A之间形成硫酯中间体。Uba4和MOCS3之间的功能相似性进一步证明了atp依赖性蛋白偶联和atp依赖性辅因子硫化之间的进化联系。
Because of mechanistic parallels in the activation of ubiquitin and the biosynthesis of several sulfur-containing cofactors, we have characterized the human Urm1 and Saccharomyces cerevisiae Uba4 proteins, which are very similar in sequence to MOCS2A and MOCS3, respectively, two proteins essential for the biosynthesis of the molybdenum cofactor (Moco) in humans. Phylogenetic analyses of MOCS3 homologues showed that Uba4 is the MOCS3 homologue in yeast and thus the only remaining protein of the Moco biosynthetic pathway in this organism. Because of the high levels of sequence identity of human MOCS3 and yeast Uba4, we purified Uba4 and characterized the catalytic activity of the protein in detail. We demonstrate that the C-terminal domain of Uba4, like MOCS3, has rhodanese activity and is able to transfer the sulfur from thiosulfate to cyanide in vitro. In addition, we were able to copurify stable heterotetrameric complexes of Uba4 with both human Urm1 and MOCS2A. The N-terminal domain of Uba4 catalyzes the activation of either MOCS2A or Urm1 by formation of an acyl-adenylate bond. After adenylation, persulfurated Uba4 was able to form a thiocarboxylate group at the C-terminal glycine of either Urm1 or MOCS2A. The formation of a thioester intermediate between Uba4 and Urm1 or MOCS2A was not observed. The functional similarities between Uba4 and MOCS3 further demonstrate the evolutionary link between ATP-dependent protein conjugation and ATP-dependent cofactor sulfuration.