Studying the Lysine Acetylation of Malate Dehydrogenase.
Studying the Lysine Acetylation of Malate Dehydrogenase.
复制标题
DOI：
10.1016/j.jmb.2017.03.027
复制
发表时间：
2017-05-05
影响因子：
5.6
通讯作者：
Fan C
中科院分区：
文献类型：
--
作者：
Venkat S;Gregory C;Sturges J;Gan Q;Fan C
蛋白质乙酰化在许多生物学过程中起着重要作用。苹果酸脱氢酶（MDH）是三羧酸（TCA）循环中的关键酶，通过蛋白质组学研究已确定其在细菌中被乙酰化，但尚未有进一步的表征报道。研究蛋白质乙酰化的一个挑战是在特定位置获得纯乙酰化的蛋白质。在这里，我们应用遗传密码扩展策略，将Nε-乙酰赖氨酸定点整合到MDH中。赖氨酸残基的乙酰化可提高MDH的酶活。大肠杆菌脱乙酰酶CobB能使乙酰化MDH脱乙酰，而大肠杆菌脱乙酰酶CobB能使乙酰化MDH脱乙酰。coli乙酰转移酶YfiQ不能有效地乙酰化MDH。我们的研究结果还表明，乙酰辅酶A或乙酰磷酸可以在体外化学乙酰化MDH。此外，MDH的乙酰化水平被证明是受碳源的生长培养基。
Protein acetylation plays important roles in many biological processes. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid (TCA) cycle, has been identified to be acetylated in bacteria by proteomic studies, but no further characterization has been reported. One challenge for studying protein acetylation is to get purely acetylated proteins at specific positions. Here we applied the genetic code expansion strategy to site-specifically incorporate Nε-acetyllysine into MDH. The acetylation of lysine residues in MDH could enhance its enzyme activity. The Escherichia coli deacetylase CobB could deacetylate acetylated MDH, while the E. coli acetyltransferase YfiQ cannot acetylate MDH efficiently. Our results also demonstrated that acetyl-CoA or acetyl-phosphate could acetylate MDH chemically in vitro. Furthermore, the acetylation level of MDH was shown to be affected by carbon sources in the growth medium.