The nature of the catalytic domain of 2′-5′-oligoadenylate synthetases
The nature of the catalytic domain of 2′-5′-oligoadenylate synthetases
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DOI：
10.1074/jbc.274.36.25535
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发表时间：
1999-09-03
影响因子：
4.8
通讯作者：
Sen, GC
中科院分区：
文献类型：
--
作者：
Sarkar, SN;Ghosh, A;Sen, GC
2‘-5’-寡腺苷(2-5(A))合成酶是一类干扰素诱导的酶，由双链RNA激活。为了理解为什么它们不同于其他DNA和RNA聚合酶，它们催化2‘-5’而不是3‘-5’磷酸二酯键的形成，我们使用分子模拟来比较DNA聚合酶β的催化结构域(Polbeta)和2-5(A)合成酶的P69同工酶的一个区域的结构。虽然P69的初级序列同源性很低，但像polB一样，P69在这个区域可以呈现出αββαβ结构。此外，P69的三个Asp残基对应于polβ的三个催化位点Asp残基的突变使该酶失活，而不影响其底物和激活剂的结合能力，进一步支持了该区域是P69的催化域的概念。该结构域在所有2-5(A)合成酶同工酶中高度保守，生化和突变研究表明，P69蛋白的二聚化是其酶活性所必需的。然而，含有野生型亚基和非活性催化结构域突变亚基的二聚体也是活性的。异源二聚体的催化速率是野生型同源二聚体的一半，尽管这两种蛋白质与双链RNA和ATP结合得同样好。
2'-5'-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induced enzymes that are activated by double-stranded RNA. To understand why, unlike other DNA and RNA polymerases, they catalyze 2'-5' instead of 3'-5' phosphodiester bond formation, we used molecular modeling to compare the structure of the catalytic domain of DNA polymerase beta (pol beta) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary sequence identity is low, like pol beta, P69 can assume an alpha beta beta alpha beta beta beta structure in this region. Moreover, mutation of the three Asp residues of P69, which correspond to the three catalytic site Asp residues of pol beta, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes, Biochemical and mutational studies demonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of the heterodimer was half of that of the wild type homodimer, although the two proteins bound double-stranded RNA and ATP equally well.