Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2′-O methyltransferase activity in dengue virus
Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2′-O methyltransferase activity in dengue virus
复制标题
DOI：
10.1016/j.virol.2010.03.011
复制
发表时间：
2010-06-20
期刊：
影响因子：
3.7
通讯作者：
Lim, Siew Pheng
中科院分区：
文献类型：
--
作者：
Chung, Ka Yan;Dong, Hongping;Lim, Siew Pheng
来自黄病毒属的甲基转移酶 (MTase) 编码 1 型 (m(7)GpppNm) 帽结构形成所需的 N-7 和 2'-O 活性。我们进行了动力学研究以了解其进行性 N-7 和 2'-O 甲基化的机制。 N-7 到 2'-O 的连续甲基化通过随机 bi bi 和持续机制发生，不涉及酶-RNA 解离。稳态动力学参数分析表明，N-7 先于 2'-O 甲基化，因为它更快地翻转 RNA (k(cat))，从而导致催化效率提高 2.4 倍。两个反应中 S-腺苷甲硫氨酸 (AdoMet) 的米氏常数比各自 RNA 底物的米氏常数低约 10 倍，表明甲基化酶反应中的限速步骤与 RNA 模板相关。在长病毒RNA序列的背景下，与S-腺苷-高半胱氨酸相比，sinefungin对抗登革热N-7和2'-O MTase活性的效力分别高出约60倍和12倍，IC50值分别为30和41 nM。 (C) 2010 Elsevier Inc. 保留所有权利。
Methyltransferases (MTases) from the genus Flavivirus encode both N-7 and 2'-O activities needed for type 1 (m(7)GpppNm) cap structure formation. We performed kinetic studies to understand the mechanisms of its progressive N-7 and 2'-O methylations. Sequential N-7 to 2'-O methylation occurred via a random bi bi and processive mechanism that does not involve enzyme-RNA dissociation. Analyses of steady state kinetic parameters showed that N-7 precedes 2'-O methylation as it turnovers RNA faster (k(cat)) resulting in 2.4-fold higher catalytic efficiency. Michaelis constants for S-adenosyl-methionine (AdoMet) in both reactions were about 10-fold lower than for their respective RNA substrates, suggesting that the rate-limiting steps in methylase reactions were associated with RNA templates. In the context of long viral RNA sequences, and compared to S-adenosyl-homocysteine, sinefungin was about 60- and 12-folds more potent against dengue N-7 and 2'-O MTase activity, exhibiting IC50 values of 30 and 41 nM, respectively. (C) 2010 Elsevier Inc. All rights reserved.