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首页> 外文期刊>Protein Science: A Publication of the Protein Society >Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase
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Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase

机译：功能齐全的黄病毒甲基转移酶的折叠表明，黄病毒甲基转移酶可同时纯化S-腺苷甲硫氨酸而不是S-腺苷高半胱氨酸

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摘要

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 angstrom is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.
机译：黄病毒RNA的甲基化对其在感染宿主细胞中的稳定性和翻译至关重要。这种甲基化是由黄病毒甲基转移酶（MTase）介导的，其通过使用S-腺苷-1-甲硫氨酸（SAM）作为甲基供体来甲基化病毒RNA帽的N7和2-O位置。在本报告中，我们证明与先前假定的反应副产物S-腺苷-1-同型半胱氨酸相反，SAM与登革热（DNV）和在大肠杆菌中产生的西尼罗河病毒MTases（E.大肠杆菌）。该内源性SAM可以通过MTase蛋白的变性和重折叠来去除。重新折叠的DNV血清型3（DNV3）的MTase表现出与天然酶相当的甲基化活性，其2.1埃的晶体结构几乎与天然MTase相同。我们通过等温滴定量热法表征了Sinefungin（SIN）（先前描述的SAM模拟MTase功能抑制剂）与天然和折叠的DNV3 MTase的结合，并发现SIN与折叠的MTase结合的亲和力超过SIN的16倍与天然纯化的MTase结合。此外，我们显示SAM还与其他黄病毒MTases共纯化，表明通过重折叠进行纯化可能是研究黄病毒MTase抑制的普遍适用工具。

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