Avrahami Emanuel M, Levi Shahar, Zajfman Eyal, Regev Clil, Ben-David Oshrit, Arbely Eyal
Department of Life Sciences , Ben-Gurion University of the Negev , Beer-Sheva 8410501 , Israel.
Department of Chemistry and The National Institute for Biotechnology in the Negev , Ben-Gurion University of the Negev , Beer-Sheva 8410501 , Israel.
ACS Synth Biol. 2018 Oct 19;7(10):2348-2354. doi: 10.1021/acssynbio.8b00314. Epub 2018 Sep 18.
Lysine deacetylases (KDACs) are enzymes that catalyze the hydrolysis of acyl groups from acyl-lysine residues. The recent identification of thousands of putative acylation sites, including specific acetylation sites, created an urgent need for biochemical methodologies aimed at better characterizing KDAC-substrate specificity and evaluating KDACs activity. To address this need, we utilized genetic code expansion technology to coexpress site-specifically acylated substrates with mammalian KDACs, and study substrate recognition and deacylase activity in live Escherichia coli. In this system the bacterial cell serves as a "biological test tube" in which the incubation of a single mammalian KDAC and a potential peptide or full-length acylated substrate transpires. We report novel deacetylation activities of Zn-dependent deacetylases and sirtuins in bacteria. We also measure the deacylation of propionyl-, butyryl-, and crotonyl-lysine, as well as novel deacetylation of Lys310-acetylated RelA by SIRT3, SIRT5, SIRT6, and HDAC8. This study highlights the importance of native interactions to KDAC-substrate recognition and deacylase activity.
赖氨酸脱乙酰酶（KDACs）是一类催化从酰基赖氨酸残基上水解酰基的酶。最近发现了数千个潜在的酰化位点，包括特定的乙酰化位点，这使得迫切需要生化方法来更好地表征KDAC-底物特异性并评估KDACs的活性。为满足这一需求，我们利用遗传密码扩展技术将位点特异性酰化的底物与哺乳动物KDACs共表达，并在活的大肠杆菌中研究底物识别和脱酰酶活性。在这个系统中，细菌细胞充当一个“生物试管”，单个哺乳动物KDAC与潜在的肽或全长酰化底物在其中进行孵育。我们报道了细菌中锌依赖性脱乙酰酶和去乙酰化酶的新型脱乙酰化活性。我们还测量了丙酰基、丁酰基和巴豆酰基赖氨酸的脱酰化，以及SIRT3、SIRT5、SIRT6和HDAC8对Lys310-乙酰化RelA的新型脱乙酰化。这项研究强调了天然相互作用对KDAC-底物识别和脱酰酶活性的重要性。
