Autoacetylation of the Histone Acetyltransferase Rtt109
Autoacetylation of the Histone Acetyltransferase Rtt109
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DOI：
10.1074/jbc.m111.251579
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发表时间：
2011-07-15
影响因子：
4.8
通讯作者：
Denu, John M.
中科院分区：
文献类型：
--
作者：
Albaugh, Brittany N.;Arnold, Kevin M.;Denu, John M.
Rtt 109是一种酵母组蛋白乙酰转移酶（HAT），与组蛋白伴侣Asf 1和Vps 75结合，使H3 K56、H3 K9和H3 K27乙酰化，在DNA复制和维持基因组完整性中起重要作用。最近，Rtt 109的质谱和结构研究表明，活性位点残基Lys-290被乙酰化。然而，这种修饰的功能作用以及乙酰基如何添加到Lys-290上尚不清楚。在这里，我们研究了Lys-290乙酰化的机制，发现Rtt 109催化Lys-290的分子内自动乙酰化，其速度比H3乙酰化慢200倍。通过与沉默调节蛋白脱乙酰酶反应制备脱乙酰化的Rtt 109，产生具有可忽略的HAT活性的酶。Rtt 109的自动乙酰化恢复了全部HAT活性，表明自动乙酰化是HAT活性所必需的，并且是完全可逆的过程。为了剖析活化机制，用Rtt 109-Vps 75复合物的Lys-290变体进行生物化学和动力学分析。我们发现Lys-290的自动乙酰化增加了对乙酰辅酶A的结合亲和力，并提高了乙酰转移到组蛋白底物上的速率。这项研究是第一次详细调查的HAT酶调节的单网站分子内自动乙酰化。
Rtt109 is a yeast histone acetyltransferase (HAT) that associates with histone chaperones Asf1 and Vps75 to acetylate H3K56, H3K9, and H3K27 and is important in DNA replication and maintaining genomic integrity. Recently, mass spectrometry and structural studies of Rtt109 have shown that active site residue Lys-290 is acetylated. However, the functional role of this modification and how the acetyl group is added to Lys-290 was unclear. Here, we examined the mechanism of Lys-290 acetylation and found that Rtt109 catalyzes intramolecular autoacetylation of Lys-290 similar to 200-times slower than H3 acetylation. Deacetylated Rtt109 was prepared by reacting with a sirtuin protein deacetylase, producing an enzyme with negligible HAT activity. Autoacetylation of Rtt109 restored full HAT activity, indicating that autoacetylation is necessary for HAT activity and is a fully reversible process. To dissect the mechanism of activation, biochemical, and kinetic analyses were performed with Lys-290 variants of the Rtt109-Vps75 complex. We found that autoacetylation of Lys-290 increases the binding affinity for acetyl-CoA and enhances the rate of acetyl-transfer onto histone substrates. This study represents the first detailed investigation of a HAT enzyme regulated by single-site intramolecular autoacetylation.