Batra Monu, Sharma Rajesh, Chandra Vemika, Aggarwal Megha, Agarwal Uday, Gupta Pawan, Singh Rajesh Pratap, Tomar Shailly
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India.
Council of Scientific and Industrial Research-Institute of Microbial Technology, Chandigarh 160036, India.
Int J Biol Macromol. 2015;77:168-80. doi: 10.1016/j.ijbiomac.2015.03.023. Epub 2015 Mar 21.
Protein methyltransferase (CheR) catalyzes the methylation of the cytosolic domain of the membrane bound chemotaxis receptors, and plays a pivotal role in the chemotactic signal transduction pathway in bacteria. Crystal structure of CheR is available only from the gram-negative bacterium Salmonella typhimurium (StCheR), which contain a catalytic C-terminal domain, encompassing a β-subdomain, connected via a linker to the N-terminal domain. The structural-functional similitude between CheR of the gram-negative and the gram-positive bacteria remains obscure. We investigated CheR, from a gram-positive bacterium, Bacillus subtilis (BsCheR), and have identified the functional roles of its N-terminal domain, by using the in silico molecular modeling and docking approach along with mass spectrophotometry and sequence analysis. The structural studies established that the N-terminal domain directly bound to S-Adenosyl-l-homocysteine (SAH). Structural and sequence analyses revealed that the α2 helix of the N-terminal domain was involved in the recognition of the methylation site of the chemotactic receptor. Additionally, immunoblot analysis showed that the purified BsCheR was phosphorylated. Further, mass spectrometry studies detected the phosphorylation at Thr3 position in the N-terminal domain of BsCheR. Phosphorylation of BsCheR suggested a regulatory role of the N-terminal domain, analogous to its antagonistic enzyme, the chemotaxis-specific methylesterase (CheB).
蛋白质甲基转移酶（CheR）催化膜结合趋化性受体胞质结构域的甲基化，在细菌的趋化信号转导途径中起关键作用。目前仅获得了革兰氏阴性菌鼠伤寒沙门氏菌（StCheR）的CheR晶体结构，其包含一个催化性C端结构域，该结构域包含一个β亚结构域，通过一个连接子与N端结构域相连。革兰氏阴性菌和革兰氏阳性菌的CheR之间的结构功能相似性仍不清楚。我们研究了革兰氏阳性菌枯草芽孢杆菌（BsCheR）的CheR，并通过计算机分子建模和对接方法以及质谱分析和序列分析确定了其N端结构域的功能作用。结构研究表明，N端结构域直接与S-腺苷-L-高半胱氨酸（SAH）结合。结构和序列分析表明，N端结构域的α2螺旋参与趋化性受体甲基化位点的识别。此外，免疫印迹分析表明纯化的BsCheR被磷酸化。进一步的质谱研究检测到BsCheR的N端结构域中Thr3位点的磷酸化。BsCheR的磷酸化表明N端结构域具有调节作用，类似于其拮抗酶趋化性特异性甲基酯酶（CheB）。
