Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53715, USA.
J Biol Chem. 2011 Jul 15;286(28):24694-701. doi: 10.1074/jbc.M111.251579. Epub 2011 May 23.
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 ∼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.
Rtt109 是一种酵母组蛋白乙酰转移酶 (HAT)，它与组蛋白伴侣 Asf1 和 Vps75 结合，乙酰化 H3K56、H3K9 和 H3K27，在 DNA 复制和维持基因组完整性方面很重要。最近，对 Rtt109 的质谱分析和结构研究表明，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被添加到 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 Rtt109 催化 Lys-290 的分子内自动乙酰化比 H3 乙酰化慢约 200 倍。通过与一种 sirtuin 蛋白脱乙酰酶反应制备了去乙酰化的 Rtt109，产生了一种 HAT 活性可忽略不计的酶。Rtt109 的自动乙酰化恢复了完整的 HAT 活性，表明自动乙酰化对于 HAT 活性是必要的，并且是一个完全可逆的过程。为了剖析激活机制，我们对 Rtt109-Vps75 复合物的 Lys-290 变体进行了生化和动力学分析。我们发现 Lys-290 的自动乙酰化增加了与乙酰辅酶 A 的结合亲和力，并增强了向组蛋白底物转移乙酰基的速率。这项研究代表了对受单个分子内自动乙酰化调节的 HAT 酶的首次详细研究。
