Higman M A, Bourgeois N, Niles E G
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
J Biol Chem. 1992 Aug 15;267(23):16430-7.
Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.
构建了能够表达痘苗病毒mRNA加帽酶大亚基和小亚基的质粒载体，并用于转化大肠杆菌。确定了以可溶形式诱导二聚体酶或单个亚基的条件，并将加帽酶纯化至接近均一。对细菌中的加帽酶进行蛋白水解产生了一种60 kDa的产物，先前已证明其具有mRNA三磷酸酶和鸟苷转移酶活性（舒曼，S.（1990年）《生物化学杂志》265，11960 - 11966），通过氨基酸序列分析分离出该产物，结果表明它源自D1R的NH2末端。单独检测时，单个亚基缺乏甲基转移酶活性。然而，将D1R和D12L亚基混合可使甲基转移酶活性得以重建，并且这种活性的出现伴随着亚基的结合。相比之下，将D12L亚基与D1R - 60K蛋白水解片段混合未能产生甲基转移酶活性，也未导致这两种蛋白质发生物理结合。这些结果表明，甲基转移酶活性位点需要D12L亚基与D1R亚基的羧基末端部分同时存在。此外，由于mRNA三磷酸酶和鸟苷转移酶活性位点位于D1R亚基的NH2末端结构域，而甲基转移酶活性存在于该亚基的羧基末端部分和D12L中，因此该酶中必定至少存在两个独立的活性位点。
