STRUCTURE STUDIES OF MIMIVIRUS CAPPING ENZYMES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Mimi病毒是一种新近发现的多食棘阿米巴寄生虫，它有一个非常大的基因组(1.2Mb)，编码911个预测蛋白。拟态病毒蛋白质组包含一系列酶，这些酶被认为在DNA和RNA新陈代谢中发挥作用。Mimi病毒似乎编码自己的mRNA合成机制，包括一个多亚单位RNA聚合酶和一个可能的RNA封闭酶，类似于痘苗病毒D1蛋白。这些特征突出了模拟病毒基因表达策略将具有痘病毒样味道的可能性。因此，我们检查了模拟病毒封盖酶的生化特性，以及它与牛痘封盖机制的相似或不同程度。我们已经纯化、检测和结晶了三功能模拟病毒mRNA封帽酶的残基(1-237)，该酶包含一个最小的自主三磷酸酶结构域。预测的四种金属结合谷氨酸(Glu37、Glu39、Glu212、Gl214)中的每一种丙氨酸替换都会取消MimiCE的NTP磷酸水解酶活性，从而证实了它是依赖金属的病毒/真菌/原虫三磷酸酶家族的成员。因此，模拟病毒封闭酶的小三磷酸酶结构域(我们可以从细菌中纯化50毫克量)为研究病毒金属依赖的RNA三磷酸酶蛋白提供了一个容易处理的靶标。基于痘病毒封端酶和模拟病毒封端酶之间的广泛相似性，在模拟病毒封端三磷酸酶的研究中获得的观察结果应该适用于整个痘病毒，因此在结构促进药物设计中有很大的用处。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mimivirus is a recently identified parasite of Acanthamoeba polyphaga that has an extraordinarily large genome (1.2 Mb) encoding 911 predicted proteins. The mimivirus proteome embraces a slew of enzymes with imputed functions in DNA and RNA metabolism. Mimivirus appears to encode its own mRNA synthetic machinery, which includes a multisubunit RNA polymerase and a putative RNA capping enzyme that resembles the vaccinia virus D1 protein. These features highlight the likelihood that the mimivirus gene expression strategy will have a poxvirus-like flavor. We therefore examined the biochemical properties of the mimivirus capping enzyme and the extent to which it resembles or differs from the vaccinia capping machinery. We have purified, assayed, and crystallized residues (1-237) of the tri-functional mimivirus mRNA capping enzyme which comprises a minimal autonomous triphosphatase domain. Alanine substitutions at each of the four predicted metal-binding glutamates (Glu37, Glu39, Glu212, Gl214) abolished the NTP phosphohydrolase activity of MimiCE, thereby verifying its membership in the metal-dependent viral/fungal/protozoal triphosphatase family. Thus, the small triphosphatase domain of the mimivirus capping enzyme (which we can purify from bacteria in 50 mg quantities) presents a tractable target for the investigation of viral metal-dependent RNA triphosphatase proteins. Based on extensive similarities between poxvirus capping enzymes and that of mimivirus, the observations gained in the study of the mimivirus capping triphosphatase should be applicable to poxvirusus at large and, therefore, of great use in structure-facilitated drug design.