古菌蛋白质甲基转移酶aKMT的催化机理及生理功能

Protein lysine methylation occurs not only in Bacteria and Eukarya, but also in Archaea. Extensive protein methylation has been found in hyperthermophilic crenarchaea. Methylation of lysine residues has been suggested to enhance the thermal stability of proteins or reduce their susceptibility to denaturation and aggregation. Our laboratory has recently isolated a protein lysine methyltransferase, termed aKMT, from a hyperthermophilic archaeon of the genus Sulfolobus. The aKMT protein is highly conserved among crenarchaea, shows a broad substrate specificity and is believed to be responsible for methylation of at least 90% of the methylated proteins in Sulfolobus. In this project, we will undertake the following tasks: (i) to determine the molecular mechanisms of substrate binding and subsequent methyl transfer by aKMT, (ii) to determine the substrate spectrum and the preferential target sequences or structures of aKMT, and (iii) to understand the physiological functions of protein methylation in Sulfolobus islandicus by using a combination of biochemical, genetic and structural biological approaches. The results of this project will likely shed light on the evolution of the process of protein methylation.

蛋白质赖氨酸甲基化修饰不仅存在于细菌和真核生物中，也存在于古菌中。在极端嗜热泉古菌中，很多蛋白质被甲基化修饰。这种翻译后修饰被认为有可能增加蛋白质的热稳定性、使蛋白质不易变性或聚集。最近，本实验室从极端嗜热泉古菌-硫化叶菌中分离纯化了蛋白质赖氨酸甲基转移酶aKMT。这是目前已知的第一个泉古菌蛋白质甲基转移酶。aKMT在泉古菌中高度保守，具有宽泛的底物特异性。据估计，该酶至少负责硫化叶菌中90%以上的蛋白质甲基化修饰。本项目拟以冰岛硫化叶菌为模式，采用生物化学、遗传学、结构生物学等手段，解析aKMT结合底物蛋白质、催化其甲基化的分子机制，确定aKMT的底物谱及识别序列或结构，探讨蛋白质甲基化修饰的生理意义，丰富关于蛋白质甲基化修饰过程进化规律的认识。

在极端嗜热泉古菌中，很多蛋白质被甲基化修饰。本项目以冰岛硫化叶菌蛋白质赖氨酸甲基转移酶aKMT为切入点，解析aKMT结合底物蛋白质、催化其甲基化的分子机制，确定aKMT的底物谱及识别序列或结构，探讨蛋白质甲基化修饰的生理意义。本项目的主要成果如下：（1）解析了硫化叶菌aKMT-SAM以及aKMT-SAH复合物的晶体结构，发现aKMT的N端区域及SAM结合口袋对aKMT活性很重要；（2）aKMT缺失不致死，突变株（ΔaKMT）在非最适生长条件（贫瘠培养基，65oC）下的生长速率慢于野生株；比较转录组的结果显示，只有少数基因的表达在ΔaKMT中发生了2倍以上的变化；（3）修饰组学分析结果显示aKMT至少参与661个蛋白的修饰，修饰没有明显的序列及二级结构偏好性；（4）染色质蛋白Cren7与Sis7的甲基化修饰由aKMT催化，而核酸结合蛋白Sis10b的甲基化修饰则与aKMT无关；（5）Sis10b的K16位不存在乙酰化修饰，只存在甲基化修饰，该修饰不具有全局性基因转录调控作用。本项目的研究结果丰富了对古菌蛋白质甲基化修饰的分子机制及生理功能的理解。