Liu Li
Department of Microbiology and Etiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.
Cell Biochem Funct. 2006 Mar-Apr;24(2):95-102. doi: 10.1002/cbf.1292.
Mouse capping enzyme (Mce1) consists of two functional domains: the amino-terminal triphosphatase domain and the carboxyl-terminal guanylyltransferase (GTase) domain. The bifunctional Mce1 gene encodes 597 a.a. with a molecular weight approximately 68 kDa. Mce1 cDNA is located on chromosome 4A4 approximately 4A5 and is composed of 17 exons. To functionally characterize the C-terminus of Mce1, we generated four truncated proteins with 12, 30, 37, or 60 a.a. deletions from the C-terminus of either the wild type (Mce1) or the isolated GTase domain (211-597), respectively. Plasmid shuffling experiment with Saccharomyces cerevisiae GTase subunit gene CEG1 null mutant demonstrated that deletion mutants 211-567 and 211-585 were able to support cell viability in the presence of 5-fluoroorotic acid, whereas 211-537 and 211-560 were not. Consistent with the yeast genetic study, both 211-567 and 211-585 had significant GTase activity in vitro, while 211-537 and 211-560 that were only detected in the insoluble fraction in the bacterial expression system, were completely inactive. Overall, both in vivo and in vitro studies indicate that the functional domain of Mce1 is between a.a. 211 and 567, and the heptapeptide sequence between 561 and 567 may play an important role in the enzyme activity.
小鼠加帽酶（Mce1）由两个功能结构域组成：氨基末端三磷酸酶结构域和羧基末端鸟苷酰转移酶（GTase）结构域。双功能Mce1基因编码597个氨基酸，分子量约为68 kDa。Mce1 cDNA位于4A4染色体上大约4A5的位置，由17个外显子组成。为了从功能上表征Mce1的C末端，我们分别从野生型（Mce1）或分离的GTase结构域（211 - 597）的C末端产生了四种截短蛋白，缺失了12、30、37或60个氨基酸。用酿酒酵母GTase亚基基因CEG1缺失突变体进行的质粒洗牌实验表明，缺失突变体211 - 567和211 - 585在5 - 氟乳清酸存在的情况下能够支持细胞活力，而211 - 537和211 - 560则不能。与酵母遗传学研究一致，211 - 567和211 - 585在体外都具有显著的GTase活性，而仅在细菌表达系统的不溶性部分中检测到的211 - 537和211 - 560则完全无活性。总体而言，体内和体外研究均表明，Mce1的功能结构域在氨基酸211和567之间，561和567之间的七肽序列可能在酶活性中起重要作用。
